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Biomedical subjects

A Wernig

Publications and source records attributed to A Wernig.

At least 37 records · Page 2Linked to original sources
PubMed1999

Laufband (treadmill) therapy in incomplete paraplegia and tetraplegia.

Recent reports indicate that intensive training of upright walking on a treadmill (German: Laufband, LB), significantly improves walking capability in spinal cord-damaged persons. The aids provided initially are body weight support by a harness and passive setting of one or both limbs by therapists. To facilitate stepping and evoke motor automatisms, "rules of spinal locomotion" need to be applied during training. The effects of this novel locomotion therapy on patients with chronic and acute incomplete paralysis are summarized and discussed here. Many patients with chronic paralysis, still wheelchair-bound and not capable of walking without help from others, became independent and learned to walk for some distance without help. Assessment of voluntary muscle activity in resting position before and after the period of therapy often showed only small increases, rendering the involvement of complex motor reflexes (motor programs) and better utilization of remaining muscle function during walking as main sources for the improvements in locomotion. This idea is supported by electromyographic recordings. Follow-up assessments performed 0.5 to 6.5 years after discharge from the hospital show that the significant improvements achieved by LB-therapy in patients with initially chronic paralysis can be maintained under domestic surrounding. Patients with initially acute paralysis improved their walking capabilities even further. It is suggested that LB therapy may be generally applied in the motor rehabilitation of persons with acute and chronic incomplete paraplegia and tetraplegia. Its use in other diseases is discussed.

Adult↗
PubMed1999

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.

Animals↗
PubMed1998

TNF inhibits insulin induced STAT5 activation in differentiated mouse muscle cells pmi28.

Tumor necrosis factor (TNF) plays a central role in the state of insulin resistance leading to type II diabetes. We here describe the crosstalk of TNF with insulin signaling cascades in the mouse muscle cell line pmi28. TNF downregulated insulin induced insulin receptor kinase activity and insulin induced activation of the transcription factor STAT5. Our results provide evidence that the inhibitory crosstalk between TNF and insulin in skeletal muscle cells comprises an interference with the expression of STAT5 regulated genes which may play an important role in the manifestation and/or progression of insulin resistance in muscle cells.

Animals↗
PubMed1998

Polyclonal antibodies against NCAM reduce paralysis-induced axonal sprouting.

The neural cell adhesion molecule (NCAM) is upregulated in paralyzed muscles but the functional role of this upregulation is not clear. We have investigated the possible involvement of NCAM in botulinum toxin-induced axonal sprouting in mouse soleus muscles. Starting 4 days after botulinum toxin-A injection, the paralyzed muscles were exposed daily for 6 or 10 days to either rabbit polyclonal NCAM antibody or control solutions (preimmune serum or saline) or remained without further treatment. By 10 days after botulinum toxin injection, the mean number of sprouts and the mean total length of sprouts, respectively, in zinc iodide-osmium-stained preparations were 2.2 and 212 microm in untreated and control treated muscles but 1.0 and 51 microm in anti-NCAM treated muscles. By 14 days, the mean number of sprouts rose to 2.9 in untreated muscles but only 1.6 in anti-NCAM treated muscles. Macrophages/monocytes, probably originating from neighboring tissue damaged by the daily injections, were present in muscles of all groups. No T lymphocytes and no signs of muscle fiber damage were found, however, rendering antibody-mediated cytotoxic reactions as unlikely. From the blocking effects of anti-NCAM, it is concluded that NCAM plays a major role in the growth of paralysis-induced axonal sprouts.

Animals↗
PubMed1998

Maintenance of locomotor abilities following Laufband (treadmill) therapy in para- and tetraplegic persons: follow-up studies.

Recent reports indicate that walking capabilities in spinal cord damaged persons significantly improve--as compared to conventional rehabilitation therapy--after intensive training of aided (Laufband) treadmill-stepping. In the present report, follow up investigations on two collectives of spinal cord injured (sci) persons are described who had undergone (Laufband) treadmill therapy either during a period of renewed rehabilitation months or years after spinal cord injury (35 chronic patients) or during their first postacute rehabilitation period (41 acute patients). Among the initially chronic patients, 20 from 25 still wheelchair-bound before the onset of (Laufband) treadmill therapy, ie not capable of raising from the wheelchair and walking without help by other persons, became independent walkers after therapy. Assessment of voluntary muscle activity in resting position before and after the period of therapy had shown only small increases in most patients, indicating the involvement of motor automatisms and better utilisation of remaining muscle function during walking. Follow-up assessments performed 6 months to 6 1/2 years after discharge from the hospital revealed that the walking capabilities achieved by (Laufband) treadmill therapy in the 35 initially chronic patients were maintained in 31 persons, in three they had further improved, in only one it was reduced. These results indicate that the improvements achieved under clinical conditions can be maintained in every day life under domestic surroundings. From 41 initially acute patients, 15 had further improved and none had reduced his walking capability 6 months to 6 years after discharge from the hospital.

Acute Disease↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Acetylcholine receptor epsilon-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice.

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the gamma-subunit of the fetal acetylcholine receptor (AChR) by the epsilon-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the epsilon-subunit gene caused in homozygous mutant mice the persistence of gamma-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of epsilon-subunit containing AChRs is essential for normal development of skeletal muscle.

Animals↗
PubMed1996

Differential sensitivity to Mg(2+)-and tubocurarine-block of frog neuromuscular junctions in summer and winter.

Several parameters of transmitter release in neuromuscular junctions were compared in "winter' and "summer' frogs. In low Ca2+/high Mg(2+)-block, the quantal content m of endplate potentials was similar for both groups. In curarized junctions, however, endplate potentials were about twice as large in winter as in summer, and facilitation was lower and depression higher. This indicates that transmitter release is higher in winter junctions. This is not reflected in Mg(2+)-block, suggesting that strong calcium deprivation may suppress release in winter more strongly than in summer junctions.

Animals↗
PubMed1995

Functional effects of myoblast implantation into histoincompatible mice with or without immunosuppression.

1. The goals of this study were to evaluate the immunogenicity of myogenic cells (MCs) (1) immediately after implantation into regenerating muscles, and (2) following their maturation under initial immunosuppression. Implanted mouse soleus muscles were evaluated by isometric tension recordings in vitro followed by histological investigations on frozen sections. 2. Implantation of non-histocompatible myoblasts into cryodamaged soleus muscles of CBA/J mice induced immune rejection which caused large and permanent deficits in muscle force: 4-42 weeks postimplantation maximal tetanic tension was 50-60% that of intact or regenerated cryodamaged control muscles without tendency for recovery or histological signs of muscle regeneration. Specific tension (force per unit muscle weight) was also significantly reduced. 3. On frozen sections, only 62 +/- 12% of the total area was desmin-positive, that is, occupied by muscle fibres, versus 90 +/- 4% in regenerated and 92 +/- 3% in intact muscles. Also, the total number of muscle fibre profiles was significantly reduced. 4. Under immune suppression with cyclosporin A (CsA), large muscles developed within 4 weeks. Following CsA withdrawal, muscle weight and force, in addition to desmin-positive areas on cross-sections, gradually declined over several months despite continual regeneration, indicating retarded immune rejection. 5. Initial application of CsA for 8 weeks after implantation, instead of 4 weeks, did not result in better survival of the implants, nor did a higher initial dose of CsA (100 instead of 50 mg kg-1 day-1). Prolonged continuous application of a reduced dose (25 mg kg-1 day-1) did not prevent muscle wasting but caused an additional delay. 6. It is concluded that histoincompatible myoblasts are highly immunogenic and that immune rejection causes large and permanent muscle deficits indicating elimination of host muscle tissue. Initial transient immunosuppression protects the incompatible cells, but after withdrawal, prolonged immune rejection and retarded muscle wasting occur.

Animals↗
PubMed1995

Laufband therapy based on 'rules of spinal locomotion' is effective in spinal cord injured persons.

Rehabilitation of locomotion in spinal cord (s.c.) injured patients is unsatisfactory. Here we report the effects of a novel 'Laufband (LB; treadmill) therapy' based on 'rules of spinal locomotion' derived from lower vertebrates. Eighty-nine incompletely paralysed (44 chronic and 45 acute) para- and tetraplegics underwent this therapy, then were compared with 64 patients (24 chronic and 40 acute) treated conventionally. The programme consisted of daily upright walking on a motor driven LB initially with body weight support (BWS) provided by a harness and assisted limb movements by the therapists when necessary. Forty-four chronic patients with different degrees of paralysis undertook the programme for 3-20 weeks (median = 10.5), 0.5-18 years after s.c. damage. At the onset of LB therapy 33/44 patients were wheelchair-bound (no standing and/or walking without help by others) whereas at the end of therapy 25 patients (76%) had learned to walk independently, 7 patients with help [corrected]. Only 1 subject did not improve. It was striking that voluntary muscle activity in the resting position was still low in several patients who had gained walking capability. Eleven patients who could already walk before LB therapy improved in speed and endurance. Of the 44 patients, six were capable of staircase walking before LB therapy compared with 34 afterwards. In order to validate the apparent superiority of LB therapy two types of comparisons were performed. In a 'temporal' control 12 spastic paretic patients, still wheelchair-bound after the period of postacute conventional therapy, performed LB immediately thereafter. After completion of LB therapy nine of these patients had learned to walk without help from others.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1995

Cellular and molecular reactions in mouse muscles after myoblast implantation.

Implantation of skeletal muscle precursor cells is a potential means of cell-mediated gene therapy. One unresolved question is the degree of immunogenicity of such myoblasts. We designed the extreme situation of implanting cells of a non-histocompatible myoblast cell line into cryodamaged, but regeneration-capable, muscles of adult mice. Without immunosuppression donor cells are rejected within the first weeks. Immunosuppression with Cyclosporin A prevented invasion of T-lymphocytes and allowed differentiation of implanted myoblasts into myofibres as well as down-regulation of MHC expression. Still, withdrawal of Cyclosporin A after 4 weeks triggered lymphocyte invasion and cytotoxic cell reactions with rejection of donor tissue. Although the vast majority of muscle fibres was MHC-negative 1-4 days after Cyclosporin A withdrawal, single small desmin-positive profiles were weakly positive for donor MHC. Parallel with the increase in the number of lymphocytes, larger numbers of small and large muscle fibres expressed high levels of either donor, host or both, class I--but not class II--molecules. Surprisingly, immune reactions continued over several months, causing gradual loss of muscle tissue. Donor class I molecules persisted for more than 6 months after Cyclosporin A withdrawal, clearly indicating survival of donor muscle fibres despite ongoing rejection. Indirect evidence on the other hand suggests additional loss of host fibres, possibly caused by cytokine release from the immune cells (bystander damage). We conclude that transient treatment with Cyclosporin A induced a kind of tolerance related to the maturation and down-regulation of class I antigens in donor muscle fibres. It is suggested that the start of immune reaction following Cyclosporin A withdrawal is initiated by remaining small amounts of donor MHC molecules, possibly related to the continuous proliferation of the cell-lined-derived donor myoblasts.

Animals↗
PubMed1995

"Bystander" damage of host muscle caused by implantation of MHC-compatible myogenic cells.

Transplantation of normal myoblasts has been considered a potential therapy for muscle dystrophies. While survival of implanted cells has been described in animal experiments and in human trials, functional effects remained unclear. Here we report on survival of progenors of implanted C2nlsBAG cells in regenerating muscles but irreversible net loss in muscle tissue and contractile force. This is caused by immune rejection of implanted myoblasts despite MHC-compatibility and "bystander" damage of host muscle tissue.

Animals↗
PubMed1994

Expression pattern of M-cadherin in normal, denervated, and regenerating mouse muscles.

Following muscle damage in adult vertebrates, myofibers can be regenerated from muscle precursor cells (satellite cells). During this process, prenatal myogenesis is recapitulated to a large extent, both morphologically and molecularly. A putative morphoregulatory molecule involved in myogenesis is M-cadherin (Mcad), a calcium-dependent cell adhesion protein. The expression of Mcad was studied by immunofluorescence in regenerating, denervated, and normal mouse muscles. Our results demonstrate that Mcad is present in satellite cells in normal muscle. Enhanced staining at sites of contact between satellite cells and the parent muscle fiber suggests an additional, spatially restricted expression of Mcad in muscle fibers. Mcad positive cells in normal and denervated muscles did not incorporate bromodeoxyuridine within 24 hr after injection in vivo, indicating that Mcad is expressed on mitotically quiescent satellite cells. Neural cell adhesion molecule (NCAM) co-localized with Mcad in nearly all satellite cells in denervated muscles but rarely in intact muscles. At early stages of regeneration, Mcad was exclusively and strongly expressed in myoblasts. After fusion of myoblasts into myotubes, Mcad was down-regulated and was barely detectable on more mature myotubes surrounded by distinct basal lamina sheaths. These observations are in line with the idea that Mcad plays a crucial role in myogenesis. In intact muscle, Mcad might function as a molecular link between satellite cell and muscle fiber.

Animals↗
PubMed1994

Polyclonal antibodies against NCAM and tenascin delay endplate reinnervation.

Experiments were performed to block molecules with antibodies which are upregulated in nerve and muscle following denervation. The delay in endplate reinnervation was taken as a measure for their involvement in regeneration. Gluteus maximus muscles of 86 male CBA/J mice were hemidenervated by freezing the caudal gluteal nerve at a defined position. The degree of reinnervation was evaluated in identified endplates by repeated vital staining of ACh receptors with rhodaminated alpha-bungarotoxin and of axons with 4Di-2ASP. Normally, endplates were completely reinnervated by 13-14 days (108 endplates in seven muscles). After daily application of polyclonal antibodies against NCAM or tenascin, reinnervation was significantly delayed. Preimmune serum, rabbit immunoglobulins or saline did not show this effect. Several monoclonal antibodies against NCAM (H-28) and tenascin (576, 578, 630, 633) showed a tendency but no significant effect. It is concluded that both NCAM and tenascin, upregulated after denervation, are involved in axon guidance and/or endplate reinnervation.

Animals↗
PubMed1993

Regular and photodamage-enhanced remodelling in vitally stained frog and mouse neuromuscular junctions.

Repeated in vivo observations of vitally stained neuromuscular junctions allow direct monitoring of ongoing structural changes, although, normally occurring changes (remodelling) and those inflicted by the illumination itself (photodamage) need to be dissociated. In frog cutaneus pectoris muscles, stained in vivo with 4Di-2ASP twice within four to five weeks, growth only was observed in 14 out of 92 junctions (in 18 muscles), retraction only in 19, and both features simultaneously in 22 junctions, while 37 junctions showed no changes. The summed growth in a junction amounted to 5-42 microns, retraction to 5-52 microns, while overall changes in synaptic length were absent. This and the simultaneous occurrence of both, growth and retraction within a single junction, indicates junctional remodelling. Similar amounts of remodelling were observed in junctions illuminated for 180, 60 or 10-30 s, indicating that within this range and with the given optical system, blue light and 4Di-2ASP fluorescence were not harmful. Remodelling was not induced by the experimental procedure per se, since in in vitro preparations signs of sprouting (and retraction) were equally frequent in junctions of totally untreated muscles, in junctions vitally stained four to five weeks previously or vitally stained but not light-exposed in the same muscle. In endplates of mouse gluteus maximus muscles double stained with 4Di-2ASP and rhodamine-alpha-bungarotoxin, unusually large (> 10 microns) terminal sprouts and retraction with gain loss of ACh-receptors became prominent 9-29 days following illumination for more than 60 s. Frequently also, muscle fibre damage with local contracture and acute loss of stainability of axon terminals occurred; often followed by muscle fibre denervation. In contrast, no such changes occurred after lower illumination intensities (12% emission filter for green) or shorter exposure times (< 60 s); even at a fourth exposure performed within 53 days. Previously reported smaller changes indicating regular remodelling were not investigated here.

Animals↗