Functional and histologic effects of a free nonvascularised muscle graft implanted into a reinnervated muscle after prolonged denervation

Striated muscle atrophy and degeneration, which increase with the delay of denervation, represent two of the main causes for poor recovery following delayed nerve repair. The present study, using a rat model, tests the hypothesis that an adjunction of small, free, nonvascularised muscle grafts of contralateral healthy triceps into a chronically denervated triceps improves muscle regeneration and recovery following sciatic nerve repair delayed for 3 months. Our experiments seem to show a relative increase in mechanical properties in animals in which free muscle graft into the triceps was performed 3 weeks following nerve repair. The improvement of the regenerative process of muscles which have suffered a long period of denervation should be considered as an additional therapeutic procedure in the case of late nerve repair.     

Midgestational sciatic nerve transection in fetal sheep results in absent nerve regeneration and neurogenic muscle atrophy

In order to test whether fetal nerve healing and regeneration result in complete functional recovery, we transected the sciatic nerve at trunk level in 13 midgestational sheep fetuses. In 10 fetuses immediate microsurgical nerve coaptation was performed. The neonatal lambs were evaluated clinically, electrophysiologically, and histologically. On the transected side, the 10 surviving lambs showed a sensorimotor sciatic nerve paralysis and atrophy of the muscles innervated by the sciatic nerve. Somatosensory evoked potentials were weakly present in 5 animals and absent in 5 animals. Histologically, minimal signs of axonal regeneration, massive degeneration of the entire nerve, and a marked neurogenic muscle atrophy were found. These unexpected results differ from the findings after peripheral nerve transections in late gestational sheep fetuses and also from the classic wallerian degeneration-regeneration pattern that follows adult nerve injury. We speculate that the almost absent regenerative potential at midgestation is related to axotomy-induced neurotrophic factor deprivation during a developmental phase where the neurons are critically dependent on growth factor for survival.     

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.     

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.     

Muscle reinnervation following neonatal nerve crush. Interactive effects of glycosaminoglycans and insulin-like growth factor-I

This study shows that glycosaminoglycans promote muscle reinnervation following neonatal sciatic nerve injury. Such an effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy. In addition glycosaminoglycan treatment of lesioned rats increased insulin-like growth factor-I messenger RNA and protein in the reinnervated muscle, and insulin-like growth factor-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of nerve lesioned rats with insulin-like growth factor-I promoted muscle reinnervation and prevention of muscle fibre atrophy, higher levels of insulin-like growth factor-I in the reinnervated muscle and of insulin-like growth factor-I and insulin-like growth factor binding proteins in plasma. These data suggest that glycosaminoglycans are potent stimulants of muscle reinnervation and that their effects may be mediated by increased levels of insulin-like growth factor-I.     

BCL-2: the pendulum of the cell fate

The influence of suprathreshold electrical stimulation of the extensor and flexor carpi radialis muscles on biomechanical and functional movement parameters is compared with the effect of a standardized active repetitive training of hand and fingers. Twelve patients suffering from ischaemic lesions in the territory of the middle cerebral artery participated in the study, which was conducted using a multiple baseline design. Following a baseline phase that lasted between one and three weeks all patients received electrical muscle stimulation for 20 minutes twice daily. In a third phase the repetitive training of hand and fingers was conducted for 20 minutes twice daily. Both interventions were applied in addition to conventional occupational therapy and physiotherapy. With the exception of spasticity in hand and finger flexors, repetitive electrical muscle stimulation does not improve biomechanical or functional motor parameters of the centrally paretic hand and arm. The repetitive motor training, however, is appropriate to improve biomechanical and functional movement parameters significantly. Apart from a possible effect on the muscle cell itself, the electrical muscle stimulation is thought to represent a mainly sensory, i.e. proprioceptive, and cutaneous intervention, whereas the active motor training is characterized by a continuous sensorimotor coupling within motor centres of the brain. The underlying neurophysiological mechanisms as well as basic principles concerning the role of afferent input for motor learning and recovery are discussed.     

Effect of the menstrual cycle on gallbladder fasting volume and postprandial emptying in nulliparous young females

In order to determine the value of a reconstructive procedure in the peripheral nerve, experimental studies often evaluate the number and the diameter of myelinated nerve fibers as a parameter for the quality of regeneration. This study addresses the correlation between the number of fibers in a peripheral motor nerve after microsurgical reconstruction and the functional result, expressed as the force of the reinnervated muscle. In a total number of 24 sheep, the motor branch to the rectus femoris muscle was severed. The muscle was reinnervated either by direct neurorrhaphy or by nerve grafting, performed in three different ways (free grafting to the ipsilateral muscle, free grafting to the contralateral muscle, vascularized grafting to the ipsilateral muscle). In the final experiments, the muscle force in the reinnervated muscle was determined by supramaximal electrical stimulation. Number and diameter of myelinated nerve fibers were evaluated by computer-assisted morphometric analysis. Regression analysis of morphometric data and the muscle forces was calculated. No correlation was found between fiber numbers in the nerve graft and the maximal force. However, a positive correlation between the number of myelinated fibers in the motor branch distal to the site of coaptation and the functional result was observed in some cases. The diameter of myelinated fibers had no influence on the functional outcome.     

Schwann cells, neurotrophic factors, and peripheral nerve regeneration

The peripheral nervous system retains a considerable capacity for regeneration. However, functional recovery rarely returns to the preinjury level no matter how accurate the nerve repair is, and the more proximal the injury the worse the recovery. Among a variety of approaches being used to enhance peripheral nerve regeneration are the manipulation of Schwann cells and the use of neurotrophic factors. Such factors include, first, nerve growth factor (NGF) and the other recently identified members of the neurotrophin family, namely, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5); second, the neurokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF); and third, the transforming growth factors (TGFs)-beta and their distant relative, glial cell line-derived neurotrophic factor (GDNF). In this review article we focus on the roles in peripheral nerve regeneration of Schwann cells and of the neurotrophin family, CNTF and GDNF, and the relationship between these. Finally, we discuss what remains to be understood about the possible clinical use of neurotrophic factors.     

Noninvasive evaluation of patients with peripheral vascular disease scheduled to undergo surgery: dobutamine stress echocardiography versus myocardial perfusion imaging

The purpose of this investigation was to describe and compare two methods of recovery of atrophied skeletal muscle following short-term impaired physical mobility. An animal model was used to study morphologic adaptations of atrophied soleus and plantaris muscles to the effects of 7 days of hind-limb suspension (HS) followed by either sedentary recovery or run training during a 28-day recovery period. Significant atrophy, demonstrated by decreased mean fiber area (MFA, in square micrometers), occurred during the 7-day period of HS. During recovery, MFA returned to control values 14 days earlier in the sedentary compared with the trained groups. Runs training following short-term atrophy induced by HS did not result in the high levels of frank muscle damage and type IIC fibers previously reported following long-term (28-day) atrophy.     

Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods

This in vivo double-blind study evaluated the effect of recombinant human glial growth factor 2 (rhGGF2), a Schwann cell mitogen, on the recovery of motor function of rat sciatic nerve following crush injury. Seventy three rats were divided into three groups. Group I (n=5), sham operated; Groups II (n=34) and III (n=34) received a 100 g crush load for 2 h over a 5 mm segment of the sciatic nerve. Group III was treated with 1 mg/kg rhGGF2, via subcutaneous injection one day before nerve crush and daily for the following four days. Group II received an equivalent volume of saline as a control. Motor functional recovery was assessed by calculating the sciatic functional index (SFI) and the recovery rate of tetanic contractile force of the extensor digitorum longus (EDL) muscle. Recovery of nerve function was evident at day 11 after crush in the rhGGF2-treated animals, whereas the nerves in controls were still paralyzed. The rhGGF2-treated animals showed a significant improvement of the SFI between days 11-21 postoperatively when compared to controls. The isometric tetanic contractile force was stronger in the rhGGF2-treated group than in controls, with a significant difference at 40 to 70 Hz stimulus frequencies on day 4. Correlation analysis showed that tetanic contractile force had a linear correlation with the SFI. Histologic assessment indicated that the rhGGF2-treated animals showed less severe degeneration and earlier robust remyelination of axons than controls. The results suggest that treatment with rhGGF2 is effective in promoting nerve regeneration as seen in measurements of functional recovery and qualitative assessment of nerve morphology. The mechanism of GGF's protective effect may be related to its direct action on Schwann cells, stimulating their mitosis as well as inducing neurotrophic factors essential to neuronal maintenance and repair.     

Muscle regeneration in young and old rats: effects of motor nerve transection with and without marcaine treatment

We tested the hypothesis that after skeletal muscle regeneration in old compared with young rats damage to the motor nerve rather than damage to muscle fibers determines the magnitude of the deficits in muscle mass and maximum force (Po). The mass and Po of extensor digitorum longus (EDL) muscles of young (4 months) and old (24 months) male rats were compared two months following (i) Marcaine treatment plus simultaneous motor nerve transection, (ii) motor nerve transection alone, and (iii) Marcaine treatment alone (from data compiled previously). In both the nerve transection-only and Marcaine with nerve transection groups the recovery of mass and Po was significantly greater in young than in old rats. This is in contrast to our previous data showing that in the absence of nerve damage Marcaine-treated muscle in old rats regenerates as well as that in young rats. Our hypothesis was supported, and we conclude that impaired axonal regeneration, re-establishment of nerve-muscle contact, or both, is the critical component in the impaired regeneration of muscle grafts in old as compared with young rats.     

Application of sciatic functional index in nerve functional assessment

In order to confirm the reliability of the sciatic functional index (SFI) in the rat, SFI, muscle strength, electrophysiological, and morphometric assessments were carried out from the 10th day to the sixth month after nerve injury or repair. The results showed that the SFI has a positive correlation with all tested indices of muscle strength, electrophysiology, and morphology (r = 0.925-0.996, P < 0.01 or P < 0.001). These results indicate that the SFI is a reliable index for evaluating rat sciatic nerve regeneration and can be widely used.     

Reflex electrical stimulation for urinary incontinence

Our previous observations have shown that the electrical stimulation of muscles is prevalently reflex. One of the advantages of reflex stimulation is that it activates not only a limited number of motor units, but rather a number of muscles connected by the same reflex from a single stimulation site. Consequently, it is not necessary to place electrodes into the muscle to be activated. They can be put elsewhere provided that the same effect is obtained and that it is more convenient for the patient. Such an opportunity arises when treating urinary incontinence which involves not only the urethral sphincter but also the group of synergistic muscles of the pelvic floor. Our experiments with several patients suffering stress incontinence have shown that indirect stimulation of the levator ani with a vaginal stimulator and especially of the anal sphincter with an anal stimulator affects the urethral sphincter in the same way as direct stimulation. These findings are significant since they enable us to use external instead of implantable stimulators. External stimulation is worth trying in all cases of stress incontinence where conservative measures have failed. In our cases, the results have been very satisfactory.     

The effect of bipolar electrocautery on peripheral nerves

Although bipolar cautery was designed to minimize trauma to the central nervous system, little is known about the effects of bipolar cautery on peripheral nerve tissue. This experiment was designed to study the effect of direct bipolar cautery on a peripheral nerve and the muscles innervated by that nerve. Lewis rats (n = 200) were assigned to five different groups: control, sham, and three cautery groups (duration of either 0.5, 1.0, or 1.5 seconds). The hind limb tibial nerves were isolated in the sham group and isolated and cauterized in the cautery groups. Assessments performed at 2 hours, 2 weeks, 4 weeks, and 8 weeks postoperatively included isometric contractile function studies of both a fast- and a slow-twitch muscle, muscle weights, and nerve histology/morphometry. Significant muscle weight loss and reduced muscle function were found in the cautery groups at 2, 4, and 8 weeks (p < 0.05). Histologically, the nerves of the cautery groups showed nerve damage consistent with Sunderland's type 4 nerve injury when examined at 2 weeks and showed nerve regeneration at 4 and 8 weeks. Both the fast-twitch muscle and the shorter duration cautery were associated with faster recovery relative to the slow-twitch muscles and longer duration cautery, respectively. Bipolar cautery, as administered to rat tibial nerves in this experiment, is associated with a significant injury to the nerve and loss of function of the muscles innervated by the nerve.     

Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain

Functional assessment of rat sciatic, tibial, and peroneal nerve injuries was performed using walking track analysis. Individual walking print length (PL), toe spread (TS), and intermediate toe spread (ITS) values were measured up to 24 weeks after specific nerve transection, with or without repair. Sciatic and tibial nerve manipulation initially affected all footprint measurements, consistent with loss of intrinsic and extrinsic motor function. After sciatic repair, TS demonstrated partial recovery without any substantial recovery in PL or ITS, compared with sciatic transection values. By contrast, after tibial repair, PL values recovered dramatically, between 16 and 24 weeks, to levels not significantly different from control subjects. This was not observed after tibial transection without repair. TS recovered partially, whereas ITS recovered to control levels by 20 weeks after tibial repair. Peroneal transection resulted in multiple contractures, rendering this group unmeasurable at 4 weeks. After peroneal repair, only the PL reflected significant loss of function at 2 weeks, recovering to control values by 8 weeks. Manual TS measurements in nonwalking rats did not reflect functional nerve regeneration. Thus, individual PL measurements alone can be used to characterize functional recovery after tibial and peroneal nerve injury, whereas TS reflected recovery after sciatic nerve injury.     

Skeletal muscle regeneration during aging and after long-term denervation

Data accumulated in recent years strongly suggest that the basis for at least part of the muscle atrophy seen in old age is related to the diminution of motor innervation in normal muscle and a decreased effectiveness of reinnervation of regenerating muscle fibers. Thus, attempts to stabilize reverse the decline of the skeletal musculature during aging must take into account both the effects of aging on the peripheral nervous system and the presence of populations of denervated muscle fibers in the aging muscles. Of considerable importance is the question of how long muscle fibers in old animals can remain denervated before they begin to lose their capacity for restoration if they ultimately become reinnervated. The experimental studies reviewed here have shown that normal muscles in old animals are capable of a high degree of restoration as long as their motor nerve supply remains undamaged. After a certain period of time, denervated muscle in young animals steadily loses the capacity to restore or repair itself. To date, so little information is available on the properties of denervated muscle in old animals that meaningful comparisons cannot be made. Ultimately, ensuring that normal or injured muscle in old individuals is supplied by an effective motor innervation may be a real key to the problems of muscle loss in old age, but if such could be provided, it will be important that the old musculature, whether normal or injured, is capable of adequately responding to the innervation.     

Quantitative graphical description of portocentral gradients in hepatic gene expression by image analysis

The work loop technique was used to measure the mechanical performance in situ of the latissimus dorsi (LD) muscles of rabbits maintained under fentanyl anesthesia. After 3 wk of incrementally applied stretch the LD muscles were 36% heavier, but absolute power output (195 mW/muscle) was not significantly changed relative to that of external control muscle (206 mW). In contrast, continuous 10-Hz electrical stimulation reduced power output per kilogram of muscle >75% after 3 or 6 wk and muscle mass by 32% after 6 wk. When combined, stretch and 10-Hz electrical stimulation preserved or increased the mass of the treated muscles but failed to prevent an 80% loss in maximum muscle power. However, this combined treatment increased fatigue resistance to a greater degree than electrical stimulation alone. These stretched/stimulated muscles, therefore, are more suitable for cardiomyoplasty. Nonetheless, further work will be necessary to find an ideal training program for this surgical procedure.     

Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 micron, controls 70.8 micron; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types. This study indicates that NT-3-enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro-muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.     

Electrical activation of the expiratory muscles to restore cough

Many patients with spinal cord injury have paralysis of their expiratory muscles and, consequently, lack an effective cough. The purpose of the present study was to evaluate the utility of lower thoracic spinal cord stimulation (SCS) to activate the expiratory muscles. Studies were performed on 15 anesthetized dogs. A quadripolar stimulating electrode (Medtronic Model 3586) was inserted epidurally and on the ventral surface of the lower thoracic spinal cord. Changes in airway pressure, airflow, and internal intercostal and abdominal muscle length were monitored to assess the effects of electrical stimulation. Spinal stimulation applied at the T9-T10 spinal level provided maximal changes in airway pressure generation in preliminary experiments. All subsequent studies were therefore performed with the electrode positioned at this level. The expiratory muscles were stimulated supramaximally over a wide range of lung volumes which were expressed as the corresponding change in airway pressure. The pressure-generating capacity of the expiratory muscles was evaluated by the change in airway pressure produced by SCS during airway occlusion. Peak expiratory airflow was also monitored following release of occlusion. At FRC, deflation (-10 cm H2O) and inflation (+ 30 cm H2O), SCS resulted in positive airway pressures of 44 cm H2O +/- 4 SE, 28 cm H2O +/- 3 SE, and 82 cm H2O +/- 7 SE. The relationship between airway pressure expiratory airflow generation and lung volume was linear (slope = 1.34 +/- 0.04) over the entire vital capacity range. Our results indicate that: (1) a major portion of the expiratory muscles can be activated reproducibly and in concert by electrical stimulation, and (2) this technique may be a clinically useful method of restoring cough in spinal cord injured patients.     

Relationship between muscle fiber composition and functional capacity of back muscles in healthy subjects and patients with back pain

Back muscles are important to the stability of the lumbar spine. Muscle fiber composition may give some indication of the functional capacity of these muscles. This review explores the relationship between muscle fiber composition and functional capacity of back muscles. The reference values for the type and size of the muscle fibers found in the back musculature of healthy subjects and patients with back pain are also presented. A high percentage of type I fibers, which are larger in size than type II fibers, has been found in back muscles at the thoracic and lumbar levels. This is in accordance with the postural function of these muscles. The diameter of type II fibers is smaller in females than males, which may partly explain the lesser strength and greater endurance capacity of back muscles in females. Due to the limited amount of pertinent data, no conclusive evidence is available regarding age-related changes in muscle fiber composition in the musculature of the back. In patients with lumbar disorders, pathological changes and selective atrophy of type II fibers are seen, and these can be changed with adequate exercises. Further research is suggested to address issues related to gender, age, back pain, and exercise and their effects on the apparent back muscle fiber composition.