Introduction

The scientific literature on the aging nervous system is immense, in particular the works on the decay of cognition and mobility. However, the impact of muscle denervation on aging skeletal muscle fibers is a relatively an orphan topic. This is related in part to the difficulties in determining by molecular approaches if motoneurons release chemical neurotrophic agents to the muscle fibers of the motor units [1]. It is well known that such mechanisms contribute to neuromuscular junction development and maintenance; however, if and what chemical trophic factors influence the synchronized expression of the hundreds of nuclei belonging to a single muscle fiber remains a subject of hypotheses, while the synchronized spread of muscle action potential seems to be a more rational mechanism [1]. The conclusions of two recent reviews [2, 3] that provide a summary of what is known or hypothesized about this subject will be discussed in the following chapters. The aim of our effort is to contribute the experience of muscle anatomists, biochemists, physiologists and skeletal muscle rehabilitation specialists concerning the impact of the denervation/reinnervation processes on shaping aging muscle fibers. We will focus on our own approaches and results which are poorly recognized in the aging community. We hope to provide rational explanations for contradictions existing in the literature on the nature and effects of the aging-related decrease in muscle bulk and on the increasing “slowness” of the aging muscle.

It is well established that the muscle deterioration that occurs with aging may be combatted by coordinated nutritional approaches and physical exercise. We will thus start with the main contradiction to this statement: the world record series of Master Athletes shows an almost linear decay in muscle power that starts at age 30 years and declines to the null value at around 110 years [4]. It is worth stressing that this occurs despite the fact that these record-holding men are the best that the human species can provide in terms of genetics and capacity to train under the best possible advice. Herein, we discuss a compilation of our investigations, involving Master Athletes, active or sedentary seniors, Spinal Cord Injured (SCI) patients, and horses. Altogether, these studies highlight the idea that volitional exercise and home-based Functional Electrical Stimulation (h-bFES) are crucial for maintaining both motor neurons and muscle fibers [5].

Lessons from Master Athletes

A 1925 paper by Hill [6] stated that valuable information concerning mobility may be found in the records of sport competitions. Accordingly, we studied the performance of world record holding Master Athletes and deduced relevant hints about the pattern of changes in muscle. Our results produce trend lines which show that power decline commences at 30 years and decreases toward zero at the age of 110 years (Fig. 1). Muscle power declines in a linear fashion, and this loss of power is a rather consistent 25 % every 20 years [4]. Each world champion is the best in his area, representing just one out of billions of people today. Even these exceptional athletes (conceivably, with optimal genetic backgrounds, focused attitudes toward training and performing, and access to the best trainers) lose power drastically as they age. Thus, it appears that something in our genome dictates this decline and nothing (as of yet) has been found to prevent it. The size and power of our muscles can go up and down several times in our lifetime in accordance with our nutritional and activity statuses; however, no matter how well we may fight aging, in the end, we will lose. First conclusion: Do not expect too much from training and rehabilitation!

Fig. 1
figure 1

Age-related decline of skeletal muscle power derived from world records of running, jumping and throwing events of Master Athletes within different age classes. Lines light blue 100 m run; red 400 m run; blue long jump; gray high jump; yellow shot put, green hammer throw

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Lessons from persons with or without a lifelong history of high-level activity

To address the situation for non-exceptionally athletic persons, we collected data and compared the muscle of young (not world class) sportsmen with that of two groups of senior people, either sedentary persons or those with a lifelong history of a high activity level. Age-related decay is strongly influenced by lifestyle, in particular activity level. [2, 7, 8]. Electrophysiological and histological measurements of skeletal muscle in older adults have detected reductions in the number of motor units and the presence of fiber type groupings [9–12] that are suggestive of denervation [13–16].

In our group of sedentary seniors, the majority of muscle fibers appear to co-express both slow and fast Myosin Heavy Chain (MHC), and some of these fibers are peculiarly small and angulated in appearance—likely a result of being denervated [8]. In contrast, muscle fibers are larger in the senior amateur sportsmen, and there is a larger number of “slow fiber type groupings” in these muscles [8]. The “type grouping” is strong evidence that the muscle fibers were “denervated” and then “reinnervated” by different motoneuron axons [8, 17]. Figure 2 shows that the denervation/reinnervation process starts with a checkerboard cluster of muscle fibers. In Fig. 2a, green fluorescence indicates fast MHC type fibers while in Fig. 2b the red indicates slow MHC type fibers. Fibers that co-express both fast and slow MHC (Fig. 2c) are orange and either of normal size or atrophic (white stars). It is likely that these are fibers which have been denervated by axonal damage and then reinnervated by a single regenerated slow motor axon. Incremental summation of denervation/reinnvervation processes may occur to such a degree that whole muscle biopsies might exhibit very large, almost complete reinnervation [7]. More importantly muscle fibers co-expressing both slow and fast MHC are normal in size in the senior sportsmen and often fill in the gaps that occur between clusters of slow myofibers [8].

Fig. 2
figure 2

Denervation/reinnervation process starts with a checkerboard cluster of muscle fibers. Green fluorescence in panel a identifies fast MHC type fibers while the red in panel b slow MHC type fibers. Fibers that co-express both fast and slow MHC (circled in c) are either normal-sized or atrophic (white star). It is likely that these are fibers which have been denervated by axonal damage and then reinnervated by a single regenerated motor axon

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Consonant findings are reported in a recent study of older obese adults, showing that 5 months of resistance training enhanced skeletal muscle innervation [18]. Indirect evidence of the occurrence of background denervation with aging was reported from the electrostimulation of leg muscles in old rats [19]. Contractile activity generated by electrical stimulation eliminated age-related losses in muscle mass and reduced the deficit in force by 50 %, providing support for the hypothesis that during aging decreased contractile activity in fibers contributes to muscle weakness and even exacerbates atrophy.

Why muscle fibers are preferentially reinnervated by axons sprouting from slow motoneurons with age is a critical question. Our opinion is that this is related to the fact that slow motoneurons are activated much more often per day than fast motoneurons [20] and that their frequent firing preferentially spares them [1]. The idea that denervation occurs naturally with aging is based on evidence of reinnervation, and we may say this because in the normal muscle of young sportsmen, there are few to no type groupings [21]. In a recent review, Hepple and Rice [3] state that “ageing motor units manifest … a reduction in motor unit number secondary to motor neuron loss, fiber type grouping due to repeating cycles of denervation-reinnervation,… Regular muscle activation through habitual physical activity … can attenuate some of these changes…”. We may underscore these statements: Our conclusion is that senior sportsmen have greater myofiber diameters, a lower percentage of denervated myofibers and a higher number of slow type groupings because they were more physically active for a greater portion of their lives. It confirms that exercise has beneficial effects on age-related muscle degradation because it promotes also muscle fiber reinnervation, preferentially rescuing slow-type motoneurons. Indeed, the opposite life-style (i.e., the low level of daily activity of sedentary persons and the even lower activity of persons with mobility impairments), produces progressive muscle weakness with muscle atrophy [2, 7, 8, 17].

Thus, high-level daily activity not only maintains trophism of muscle fibers, but also trophism of slow motoneurons and/or increases their axon sprouting potentials [2, 3, 8, 18].

Lessons from sedentary elderly persons: effects and mechanisms of muscle Functional Electrical Stimulation (FES)

In addition to a progressive loss of muscle mass, aging skeletal muscle also presents with a conspicuous reduction in myofiber plasticity [2, 17, 22] and alterations in muscle-specific transcriptional mechanisms. During the aging process, protein synthetic rates decrease and an increase in protein degradation follows [23, 24], affecting characteristics of muscle fibers [25]. It is generally accepted that the failure to repair damage is a contributory cause of functional impairment with aging [26–29] and promotes the detrimental replacement of functional contractile muscle with fibrous tissue [25].

Volitional physical exercise can reverse these damaging processes [2, 3]. Interestingly, it has been shown that both acute and prolonged resistance exercise stimulate the proliferation of satellite cells in healthy sedentary elderly subjects [30–34], though blunted in elderly people. This fact may be explained by the increased levels of myostatin [35], a negative regulator of muscle mass [35, 36]. An increase in autophagy in the muscle of athletic people has been reported [37–39], suggesting that exercise may activate an important system that detoxifies muscle cells. Another major factor that is associated with physical exercise is Insulin-like Growth factor 1 (IGF-1) [40]. IGF-1 production by muscle increases after 5–10 min of moderate- to high-intensity exercise [41–43]. The evidence suggests that training and regular exercise, by modulating functional autophagy, myokines (IL-6) and IGF-1 expression, can increase muscle strength and attenuate sarcopenia [21, 37, 44]. There is strong evidence that IGF-1 secretion by muscle fibers also influences synaptogenesis in the brain [45] and at neuromuscular junctions [46, 47]. Further, agrin (along with other proteins such as MuSK, rapsyn, ChAT, Hb9, munc18, etc.) is important to denervation/reinnervation processes [48].

Unfortunately, elderly people may be unable or reluctant to adequately participate in physical exercise. We suggest that an alternative approach is functional electrical stimulation. A stimulator for neuromuscular electrical stimulation (ES) was designed, which especially suits the requirements of elderly people [49]. As detailed in Kern et al. [23], subjects were exposed to regular neuromuscular ES training for a period of 9 weeks. The outcome was an increase in muscle strength, associated with an increase of fast fibers, which are the first to respond to ES and are related to the power of skeletal muscle [23]. Because IGF-1 is one of the factors that is activated during physical exercise, we asked if ES would induce an increase in expression of IGF-1 and other components of downstream pathways. Indeed, we discovered that ES increases expression of IGF1 and of markers of both satellite cell proliferation and extracellular matrix remodeling and that it downregulates expression of proteases [23]. We also explored collagen expression and reported that there is remodeling during both volitional physical exercise and ES. Three different forms of collagen are also upregulated in electrically stimulated muscle [23], but the increase in collagen likely does not stimulate fibrosis as is shown by both morphological evidence and at the level of important fibrosis modulators, namely the increase in expression of miR29 [50].

Several longitudinal studies have shown that regular exercise is beneficial to aged population [2, 3, 12, 40, 51]. Thus, we wanted to compare the effects of regular exercise and ES on senior people. The Interreg IVa project recruited sedentary seniors with a normal lifestyle who were trained for 9 weeks with either leg press (LP) exercise [52] or ES [22, 23, 49]. Functional tests of trained subjects showed that LP and ES induced improvements in leg muscle force and of mobility [53, 54]. ES significantly increased the size of fast-type muscle fibers, together with a significant increase in the number of Pax7 and NCAM positive satellite cells. Further, no signs of muscle damage and/or inflammation were observed in muscle biopsies. Altogether the results demonstrate that physical exercise, either voluntary (LP) or induced by ES, improves the functional performance of aging muscles. However, neither training can stop the aging process as we have learned from the Master Athletes. The data show that ES is a safe home-based method able to counteract atrophy of fast-type muscle fibers, a biomarker of skeletal muscle aging in sedentary seniors [17, 22].

Lessons from persons affected by spinal cord injury

Premature aging may occur as a result of many chronic diseases, including those which involve loss of muscle innervation. One extreme case is irreversible Conus and Cauda Equina syndrome, a spinal cord injury (SCI) sequela. In this syndrome, the leg muscles may be completely and permanently disconnected from the nervous system and their fibers, thus, undergo severe atrophy and finally fibro-fatty degeneration. [55–63]. In the case of SCI with only upper motor neuron lesions, likewise those with complete transverse lesion at the thoracic level, muscle fibers below the lesion remain innervated but disused; nonetheless, their disuse induces, at most, a 50 % decrease in fiber size which remains stable for at least 20 years post-SCI [64]. Such patients exhibit a stable histochemical ATPase checkerboard pattern, and the slow-type fibers may stain more lightly [64].

Otherwise, looking at the effects of long-term denervation on human muscle in irreversible Conus and Cauda Equina syndrome (Fig. 3), four phases have been identified: (1) ultrastructural disorganization and loss of contractile response to ES (within a few months); (2) muscle atrophy (up to 2 years after SCI); (3) muscle degeneration with severe muscle atrophy (3–6 years after SCI); and (4) loss of myofibers (more than 3 years after SCI) [55–63, 65].

Fig. 3
figure 3

Permanent long-term denervation simulates premature aging in muscle. EU Program RISE: use of electrical stimulation to restore standing in paraplegics with long-term denervated degenerated muscles (Contract no. QLG5-CT-2001-02191)

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On the other hand, we have shown that home-based Functional Electrical Stimulation (h-bFES) of permanently denervated muscles stops and reverses the degeneration of skeletal muscle tissue [55–63]. Indeed, 2D Muscle Color Computer Tomography scans of thigh muscles in transverse section (Fig. 4) demonstrate the effect of h-bFES on long-term permanently denervated leg muscles. After two additional years of permanent denervation, but with FES treatment (Fig. 4g–j), the muscles began to resemble normal tissue. Indeed, the colors represent different tissues, computed on the basis of their radiodensity. These results are quite astonishing, highlighting the utility of h-bFES to greatly improve even badly degenerated denervated muscle tissue [55–63, 65]. As further evidence of the efficacy of h-bFES, Fig. 5 shows that 5 years after discontinuation of FES the recovered leg muscle deteriorates again [66–68]. It is evident, by our data, that h-bFES training (five times per week at 3 h per day) reverses atrophy and maintains microscopic and macroscopic features of the muscle fibers [56, 58, 60–63, 65]. If FES training is started within the first year following SCI, the retention of muscle is almost complete and may even rescue the ability to stand up and perform “walking in place” exercises during the second year of h-bFES [61]. If one induces many contractions daily in those muscle fibers that are otherwise destined to die, these fibers will survive and contract for up to dozens of years, but only if one stimulates them with very long, high amplitude impulses delivered by large surface electrodes designed for stimulating denervated muscles, i.e., using the patterns of stimulation developed in Vienna by Prof. Helmut Kern, Eng. Christian Hofer, Prof. Winfried Mayr and collaborators [55–65]. Commercial devices designed for electrical stimulation of long-term permanently denervated muscles that are capable of producing the needed stimulation patterns are currently available. Therefore, this excellent therapy should become accessible to more people for the preservation of skeletal muscle.

Fig. 4
figure 4

2D Color CT evidence of recovery from permanent denervation (i.e., premature muscle aging) by home-based Functional Electrical Stimulation (h-bFES). Color scans of thigh muscles before (b–e) and after 2 years (g–j) of h-bFES. Each panel Cross-sectional area and the quality of quadriceps muscles in patients starting h-bFES at different time points after denervation (b 1.2; c 1.7; d 3.2; e 5.4 years) increased after 2 years of home training (g–j, respectively). Moreover, the interstitial tissues that increased with post-denervation time (compare yellow, green and blue areas in b–e) decreased in the respective patient after 2 years of h-bFES (g–j, respectively)

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Fig. 5
figure 5

3D Color Muscle CT reconstruction of the rectus femoris: reversibility of h-bFES-induced muscle recovery in a non-compliant patient. 3D reconstructions of rectus femoris from the SCI patient at three time points. a Four years after SCI, b after 4 years of h-bFES, and c 5 years after discontinuing h-bFES training. Five years after discontinuation of FES the recovered leg muscle deteriorates again

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Research conducted over the past 50 years has demonstrated that muscle activity, not neurotrophic substances, is the most important factor in the regulation of size and of specific physiological and biochemical properties of muscle fibers. Application of this knowledge has led to considerable experimentation with chronic electrical stimulation as a possible clinical tool for the treatment of denervated muscles. Evidence accumulated from animal studies has indicated that direct electrical stimulation of denervated muscles can, to a significant extent, directly substitute reinnervation and preserve or restore the normal properties of these muscles. Appropriate stimulation parameters were critical for successful intervention, and the best results were obtained when the stimulation pattern resembled the differential firing pattern of motoneurons [1, 69, 70]. Nonetheless, even now, the vast majority of neurologists and physiatrists, who are not yet aware of those results, profess that denervated muscle cannot be maintained and certainly not regenerated [56, 63, 71]. Furthermore, based mainly on animal studies during the stage of removal of polyneural innervation in developing muscle fibers [72], there is a common sentiment that FES may hamper reinnervation despite the mounting evidence of the role of exercise in maintaining the integrity of the neuromuscular junction [73].

Contrastingly, many pioneering and recent experiments are showing the contrary. Denervated muscles, stimulated electrically for 4 days prior to reinnervation, show preserved endplate structure as well as accelerated recovery of normal function in reinnervated muscle fibers after 11 days of denervation [74]. Furthermore, electrical stimulation improves muscle reinnervation both functionally and structurally [5, 75–78].

Apparently, additional evidence is necessary to fully convince skeptics that FES is effective. A by-product of our previous studies, clinical imaging by 2D and 3D Muscle Color Computed Tomography (Fig. 6a, b) may be useful in collecting final evidence of the effectiveness of FES. In particular, this 3D methodology allows for evaluation of the effects of FES on an entire muscle [66–68]. Widely used in cardiac imaging [79], the false color approach is basically ignored in clinical imaging of skeletal muscle tissue. Our aim is to convince readers that the advantages inherent to this imaging technique offset the low risks of irradiation, in particular during follow-up of supervised trials.

Fig. 6
figure 6figure 6

2D and 3D Muscle Color CT scan. a The distribution analysis of the ranges of Hounsfield Unit in the histograms (left panels) allows for much more detailed quantitation of the changes occurring within the soft tissues of the leg (i.e., fat in yellow and muscles in red or orange, according to their density). Two months of conventional physiotherapy and electrical stimulation increased the content of low density muscle in the right leg by 2 % at the expense of the intramuscular fat and fibrous-dense connective tissue. The histograms in a refer to the muscle density on the tibialis anterior that is highlighted with black contours in all cross sections. b Density distribution and segmented areas on specific cross sections

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Readers interested in neuromuscular electrical stimulation (NMES) by surface or implantable devices for other more frequent syndromes due to SCI or stroke may find valuable information in other literature sources [80–82]. Furthermore, the debated topic of electrical stimulation and/or exercise to promote regeneration of damaged peripheral nerves is reviewed in detail in Gordon T, English AW 2016 [5].

Lessons from horse models

Finally, we would like to discuss a recent example of the effectiveness of FES for treatment of equine epaxial muscle spasm. In this example, the different types of mitochondria present in skeletal muscle fibers, either subsarcolemmal or intermyofibrillar and their differential responses to needs and activation loads of muscle fibers were explored [83–85]. Preliminary histopathologic analyses had suggested that stimulated muscles were more damaged after treatment than they were before (Sheila Schils, personal communication). More careful morphometric analyses of the same samples excluded this possibility. Indeed, only one horse presented with obvious evidence of post-FES muscle damage, whereas the other five horses, which had the same type and amount of electrical stimulation, displayed scarce or none evidence of muscle atrophy/damage in both pre- and post-stimulation biopsies [84, 85]. If one does morphometry properly, the correlation between FES and muscle damage often disappears.

Regardless, in the post-FES horse biopsies, NADH-TR staining of mitochondria showed an increase of mitochondria that are localized near the sarcolemma. It is interesting to note that in recent observations comparing young and very old (30 months) rat muscles (EDL, soleus and diaphragm) subjected to neurectomy [86], we found that the abundance of subsarcolemmal mitochondria decreases with age—even more so after 1 week of denervation, whatever the animal’s age (manuscript in preparation). Feliciano Protasi and others have shown very interesting data concerning the structural relationships of mitochondria with triads in the intermyofibrillar spaces [56, 58, 61]. They conclude that physical interactions are part of the control mechanisms that provide the needed amount of ATP to the contractile machinery [87, 88]. In muscle fibers, there are different types of mitochondria, and they respond differently to various muscle fiber needs. Mitochondrial distributions and densities are significantly changed in horse post-FES muscle biopsies. This indicates that the clinical improvements observed in these horses are related to increased muscle perfusion induced by FES stimulation [83–85]. Thus, as in human cases [55–65], FES provides clinically relevant results in the treatments of equine epaxial muscle spasm. Interesting results were also recently published on the effects of FES by an implantable device on denervated laryngeal muscle in horses. Eight-week daily electrical stimulation produced a 30 % increase in fiber diameter in comparison with an unstimulated control group, as well as a trend toward a decrease in the proportion of type 1 (slow) fibers. The data confirm that ES by an implanted device, in accordance with the use of a relatively conservative set of stimulation parameters, can reverse the muscle fiber atrophy produced by complete denervation [89].

Conclusion and perspectives

In summary, it appears that the age-related decline in muscle power is partially attributable to a loss of innervation and that this loss can be deferred by lifelong high-level activity [7, 8]. Diseases involving permanent denervation show similar, but premature, aging and much more severe muscle deterioration. Despite doubts and criticisms of related literature [80, 82], we have shown that, with appropriate protocols, h-bFES can inhibit degeneration of denervated muscle and even reverse it [61, 65]. Thus, it should be possible to defer age-related muscle decline in aging people and in others who have become unable to participate in daily physical activities. Its use will be hopefully extended to muscle reinnervation after peripheral nerve lesions. In truth, the data indicate that FES should be considered for use in critical care units, rehabilitation centers, nursing facilities and at home when patients are unable or reluctant to perform volitional physical exercise even for short periods of time.