Motoneuron regeneration accuracy and recovery of gait after femoral nerve injuries in rats.

The rat femoral nerve is a valuable model allowing studies on specificity of motor axon regeneration. Despite common use of this model, the functional consequences of femoral nerve lesions and their relationship to precision of axonal regeneration have not been evaluated. Here we assessed gait recovery after femoral nerve injuries of varying severity in adult female Wistar rats using a video-based approach, single-frame motion analysis (SFMA). After nerve crush, recovery was complete at 4 weeks after injury (99% of maximum 100% as estimated by a recovery index). Functional restoration after nerve section/suture was much slower and incomplete (84%) even 20 weeks post-surgery. A 5-mm gap between the distal and proximal nerve stumps additionally delayed recovery and worsened the outcome (68% recovery). As assessed by retrograde labeling in the same rats at 20 weeks after injury, the anatomical outcome was also dependent on lesion severity. After nerve crush, 97% of the femoral motoneurons (MNs) had axons correctly projecting only into the distal quadriceps branch of the femoral nerve. The percentage of correctly projecting MNs was only 55% and 15% after nerve suture and gap repair, respectively. As indicated by regression analyses, better functional recovery was associated with higher numbers of correctly projecting MNs and, unexpectedly, lower numbers of MNs projecting to both muscle and skin. The data show that type of nerve injury and repair profoundly influence selectivity of motor reinnervation and, in parallel, functional outcome. The results also suggest that MNs' projection patterns may influence their contribution to muscle performance. In addition to the experiments described above, we performed repeated measurements and statistical analyses to validate the SFMA. The results revealed high accuracy and reproducibility of the SFMA measurements.

Deficient functional recovery after facial nerve crush in rats is associated with restricted rearrangements of synaptic terminals in the facial nucleus.

Crush injuries of peripheral nerves typically lead to axonotmesis, axonal damage without disruption of connective tissue sheaths. Generally, human patients and experimental animals recover well after axonotmesis and the favorable outcome has been attributed to precise axonal reinnervation of the original peripheral targets. Here we assessed functionally and morphologically the long-term consequences of facial nerve axonotmesis in rats. Expectedly, we found that 5 months after crush or cryogenic nerve lesion, the numbers of motoneurons with regenerated axons and their projection pattern into the main branches of the facial nerve were similar to those in control animals suggesting precise target reinnervation. Unexpectedly, however, we found that functional recovery, estimated by vibrissal motion analysis, was incomplete at 2 months after injury and did not improve thereafter. The maximum amplitude of whisking remained substantially, by more than 30% lower than control values even 5 months after axonotmesis. Morphological analyses showed that the facial motoneurons ipsilateral to injury were innervated by lower numbers of glutamatergic terminals (-15%) and cholinergic perisomatic boutons (-26%) compared with the contralateral non-injured motoneurons. The structural deficits were correlated with functional performance of individual animals and associated with microgliosis in the facial nucleus but not with polyinnervation of muscle fibers. These results support the idea that restricted CNS plasticity and insufficient afferent inputs to motoneurons may substantially contribute to functional deficits after facial nerve injuries, possibly including pathologic conditions in humans like axonotmesis in idiopathic facial nerve (Bell's) palsy.

Functional deficits and morphological changes in the neurogenic bladder match the severity of spinal cord compression.

UNLABELLED: Following spinal cord injury (SCI), loss of spinal and supraspinal control results in desynchronisation of detrusor vesicae (parasympathicus) and external urethral sphincter (sympathicus) activity. Despite recovery of lower urinary tract function being a high priority in patients with SCI, effective treatment options are unavailable largely because mechanisms are poorly understood. PURPOSE AND METHODS: We used a clinically relevant model of thoracic SCI compression injury in adult female Wistar rats and confirmed that lesion volumes following severe injuries were significantly greater compared to moderate injuries (p < 0.05). Between 1-9 weeks, we assessed recovery of bladder function as well as return of locomotor function using the Basso, Beattie and Bresnahan (BBB) score. Bladder morphometrics and overall intramural innervation patterns, as assessed with ß-III tubulin immunohistochemistry, were also examined. RESULTS: Despite variability, bladder function was significantly worse following severe compared to moderate compression injury (p < 0.05); furthermore, the degree of bladder and locomotor dysfunction were significantly correlated (r = 0.59; p < 0.05). In addition, at 9 weeks after SCI we saw significantly greater increases in bladder dry weight (p < 0.05) and wall thickness following severe compared to moderate injury as well as increases in intramural axon density (moderate: 3× normal values; severe 5×; both p < 0.05) that also correlated with injury severity (r = 0.89). CONCLUSION: The moderate and severe compression models show consistent and correlated deficits in bladder and locomotor function, as well as in gross anatomical and histopathological changes. Increased intramural innervation may contribute to neurogenic detrusor overactivity and suggests the use of therapeutic agents which block visceromotoric efferents.

Non-invasive stimulation of the vibrissal pad improves recovery of whisking function after simultaneous lesion of the facial and infraorbital nerves in rats.

We have recently shown that manual stimulation of target muscles promotes functional recovery after transection and surgical repair to pure motor nerves (facial: whisking and blink reflex; hypoglossal: tongue position). However, following facial nerve repair, manual stimulation is detrimental if sensory afferent input is eliminated by, e.g., infraorbital nerve extirpation. To further understand the interplay between sensory input and motor recovery, we performed simultaneous cut-and-suture lesions on both the facial and the infraorbital nerves and examined whether stimulation of the sensory afferents from the vibrissae by a forced use would improve motor recovery. The efficacy of 3 treatment paradigms was assessed: removal of the contralateral vibrissae to ensure a maximal use of the ipsilateral ones (vibrissal stimulation; Group 2), manual stimulation of the ipsilateral vibrissal muscles (Group 3), and vibrissal stimulation followed by manual stimulation (Group 4). Data were compared to controls which underwent surgery but did not receive any treatment (Group 1). Four months after surgery, all three treatments significantly improved the amplitude of vibrissal whisking to 30° versus 11° in the controls of Group 1. The three treatments also reduced the degree of polyneuronal innervation of target muscle fibers to 37% versus 58% in Group 1. These findings indicate that forced vibrissal use and manual stimulation, either alone or sequentially, reduce target muscle polyinnervation and improve recovery of whisking function when both the sensory and the motor components of the trigemino-facial system regenerate.

Recovery of whisking function after manual stimulation of denervated vibrissal muscles requires brain-derived neurotrophic factor and its receptor tyrosine kinase B.

Functional recovery following facial nerve injury is poor. Neuromuscular junctions (NMJs) are "bridged" by terminal Schwann cells and numerous regenerating axonal sprouts. We have shown that this poly-innervation of NMJs can be reduced by manual stimulation (MS) with restoration of whisking function. In addition, we have recently reported that insulin-like growth factor-1 (IGF-1) is required to mediate the beneficial effects of MS. Here we extend our findings to brain derived neurotrophic factor (BDNF). We then examined the effect of MS after facial-facial anastomosis (FFA) in heterozygous mice deficient in BDNF (BDNF(+/-)) or in its receptor TrkB (TrkB(+/-)). We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive terminal Schwann cells. In intact BDNF(+/-) or TrkB(+/-) mice and their wild type (WT) littermates, there were no differences in vibrissal whisking nor in the percentage of bridged NMJ (0% in each genotype). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (27% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (11% more than intact). After FFA and handling in BDNF(+/-) or TrkB(+/-) mice, whisking amplitude was again reduced (53% and 60% lower than intact) and proportion of bridged NMJ increased (24% and 29% more than intact). However, MS failed to improve outcome in both heterozygous strains (whisking amplitude 55% and 58% lower than intact; proportion of bridged NMJ 27% and 18% more than intact). We conclude that BDNF and TRkB are required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

[Regeneration of the facial nerve in comparison to other peripheral nerves : from bench to bedside]. / Regeneration des N. facialis im Vergleich zu anderen peripheren Nerven : Aktuelles aus der Forschung für die Klinik.

Despite increasing knowledge of cellular and molecular mechanisms determining the success or failure of peripheral nerve regeneration, no effective treatments for peripheral nerve injury exist. Newly developed and validated approaches for precise numerical assessment of motor deficits have recently allowed testing of novel strategies in experimental animals. One of these approaches is the daily manual stimulation of the denervated musculature. This treatment is effective in cases of cranial nerve lesions with preservation of the sensory input (facial or hypoglossal nerve) and has the potential of direct translation in clinical settings. However, manual stimulation appears to be ineffective for the treatment of mixed peripheral nerve injuries. Generally, no long-term improvement of functional recovery is achieved by electrical stimulation in rodents. While short-term post-traumatic stimulation of the proximal nerve stump has no negative effects, direct electrical stimulation of the muscle during the period of de- and reinnervation appears to hinder muscle fibre reinnervation. Finally, experimental evidence suggests that application of peptides known as glycomimetics, which mimic functional properties of carbohydrate molecules, may provide significant benefits after injuries of mixed peripheral nerves.

Recovery of whisking function promoted by manual stimulation of the vibrissal muscles after facial nerve injury requires insulin-like growth factor 1 (IGF-1).

Recently, we showed that manual stimulation (MS) of denervated vibrissal muscles enhanced functional recovery following facial nerve cut and suture (FFA) by reducing poly-innervation at the neuro-muscular junctions (NMJ). Although the cellular correlates of poly-innervation are established, with terminal Schwann cells (TSC) processes attracting axon sprouts to "bridge" adjacent NMJ, molecular correlates are poorly understood. Since quantitative RT-PCR revealed a rapid increase of IGF-1 mRNA in denervated muscles, we examined the effect of daily MS for 2 months after FFA in IGF-1(+/-) heterozygous mice; controls were wild-type (WT) littermates including intact animals. We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive TSC. There were no differences between intact WT and IGF-1(+/-) mice for vibrissal whisking amplitude (48 degrees and 49 degrees ) or the percentage of bridged NMJ (0%). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (42% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (12% more than intact). After FFA and handling in IGF-1(+/-) mice, the pattern was similar (whisking amplitude 57% lower than intact; proportion of bridged NMJ 42% more than intact). However, MS did not improve outcome (whisking amplitude 47% lower than intact; proportion of bridged NMJ 40% more than intact). We conclude that IGF-I is required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

Manual stimulation of forearm muscles does not improve recovery of motor function after injury to a mixed peripheral nerve.

Transection and re-anastomosis of the purely motor facial nerve leads to poor functional recovery. However, we have recently shown in rat that manual stimulation (MS) of denervated vibrissal muscles reduces the number of polyinnervated motor endplates and promotes full recovery of whisking. Here, we examined whether MS of denervated rat forearm muscles would also improve recovery following transection and suture of the mixed (sensory and motor) median nerve (median-median anastomosis, MMA). Following MMA of the right median nerve, animals received no postoperative treatment, daily MS of the forearm muscles or handling only. An almost identical level of functional recovery, measured by the force of grip in grams, was reached in all animals by the sixth postoperative week and maintained till 3 months following surgery regardless of the postoperative treatment. Also, we found no differences among the groups in the degree of axonal sprouting, the extent of motor endplate polyinnervation and in the soma size of regenerated motoneurons. Taken together, we show that while MS is beneficial following motor nerve injury, combined strategies will be required for functional recovery following mixed nerve injury.

A model system for studying postnatal myogenesis with tetracycline-responsive, genetically engineered clonal myoblasts in vitro and in vivo.

The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 microg ml(-1)) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway.

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

Regenerative capacity and the number of satellite cells in soleus muscles of normal and mdx mice.

Satellite cells are potential myogenic cells that participate in repair and growth of muscle fibres. In this investigation, the change in the number of satellite cells following severe muscle damage was monitored in soleus muscle of age-matched mdx and C57Bl/10 mice. Satellite cells were identified immunohistochemically in the light microscope by their association with a recently described marker protein, M-cadherin, and their location between the muscle fibre's sarcolemma and the surrounding basal lamina. In cross-sections of untreated soleus muscle of C57Bl/10 mice at 11-14. 5 months of age, nuclei of M-cadherin positive satellite cells on average amounted to 3.4% of the total number of myonuclei. Surprisingly, significantly higher numbers of satellite cell nuclei, both in absolute numbers (mean 24+/-11 versus 40+/-11 satellite cells per section) and relative to the total number of myonuclei (5. 3%), were found in similarly aged animals in which severe muscle damage had been inflicted 3-6 months before. Cross-sectional area, muscle tissue area and myonuclei counts had recovered to control values. In untreated muscles of age-matched mdx mice satellite cell counts were not different (2.7% of myonuclei) from C57Bl/10 mice. However, regeneration showed marked deficits, as there was a loss of about 36% total cross-sectional area, about 48% total muscle fibre area and about 43% myonuclei per section compared to the untreated mdx muscles. Furthermore, the absolute number of satellite cells decreased from 20+/-11 to 12+/-8 per section. The relative number of satellite cell nuclei remained comparable to, but did not exceed, the undamaged muscles. The poor recovery of muscle and the missing post-regeneration rise in satellite cell numbers may indicate the reproductive limits of the satellite pool.

Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

The M-cadherin catenin complex interacts with microtubules in skeletal muscle cells: implications for the fusion of myoblasts.

M-cadherin, a calcium-dependent intercellular adhesion molecule, is expressed in skeletal muscle cells. Its pattern of expression, both in vivo and in cell culture as well as functional studies, have implied that M-cadherin is important for skeletal muscle development, in particular the fusion of myoblasts into myotubes. M-cadherin formed complexes with the catenins in skeletal muscle cells similar to E-cadherin in epithelial cells. This suggested that the muscle-specific function of the M-cadherin catenin complex might be mediated by additional interactions with yet unidentified cellular components, especially cytoskeletal elements. These include the microtubules which also have been implicated in the fusion process of myoblasts. Here we present evidence that the M-cadherin catenin complex interacts with microtubules in myogenic cells by using three independent experimental approaches. (1) Analysis by laser scan microscopy revealed that the destruction of microtubules by nocodazole leads to an altered cell surface distribution of M-cadherin in differentiating myogenic cells. In contrast, disruption of actin filaments had little effect on the surface distribution of M-cadherin. (2) M-cadherin antibodies coimmunoprecipitated tubulin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated epithelial cells ectopically expressing M-cadherin. Vice versa, tubulin antibodies coimmunoprecipitated M-cadherin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of control proteins, were copolymerized with tubulin from myogenic cell extracts even after repeated cycles of assembly and disassemly of tubulin. Moreover, neither M-cadherin nor E-cadherin could be found in a complex with microtubules in epithelial cells ectopically expressing M-cadherin. Our data are consistent with the idea that the interaction of M-cadherin with microtubules might be essential to keep the myoblasts aligned during fusion, a process in which both M-cadherin and microtubules have been implicated.

Ectopic skeletal muscles derived from myoblasts implanted under the skin.

We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6-78 mg, n=37) than in mice injected with primary myoblasts (1-7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture. Presence of endplates (revealed by alpha-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.

Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.

1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y-chromosome-specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin-positive cross-sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin-positive muscle cross-sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve-evoked) stimulation were equal. Endplates were found on numerous Y-positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M-cadherin, donor-derived cells contributed to the pool of satellite cells on small- and large-diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y-positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures-unlike permanent cell lines-significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe.

Impaired functional and structural recovery after muscle injury in dystrophic mdx mice.

We compared functional and structural recovery from imposed muscle injury in mdx and wild type mice to test their regenerative capacity. Soleus muscle, known to be particularly affected by the disease process, was subjected to most severe damage caused by freeze injury plus 'bystander damage'; the latter causes destruction of host muscle cells in the course of immune rejection of implanted non-histocompatible myogenic cells. Freezing/implantation was performed in mdx and control mice at two ages (4-6 months, "young' and 10-12 months, 'old' age). While recovery of muscle force in the control groups reached 77 and 88% of contralateral by 3 and 6 months, it was 60% and only 43% in mdx mice damaged at young and old age, respectively. Larger force deficits in mdx mice were due to loss of muscle tissue as measured from desmin-positive areas. Worse recovery of dystrophic muscles in general, and old muscles in particular, is interpreted to indicate pronounced exhaustion of the regenerative capacity, possibly caused by previous cycles of degeneration and regeneration.