University of Padova

Interdepartmental Research Center of Myology (cir-MYO)

Department of Experimental Biomedical Science

 

Padua Muscle Days 2006

 

October 20-21, 2006

 

Hotel Continental, Montegrotto Terme

Via Neroniane, 8 – Montegrotto Terme (Padova)

Tel +39 049 793522 - Fax +39 049 8910683 - e-mail: hotelcontinental@tin.it

 

Summaries & Abstracts

 

Friday, October 20, 2006

 

17.45    Ugo Carraro, Francesco Mazzoleni, Paolo Bernardi: Openings

18.00           Thordur Helgason, Reykjavik, Iceland: Segmentation and modeling of thigh muscles, bones, veins and arteries

18.40           Giuseppe Vita, Messina, Italy: VEGF gene transfer using adeno-associated viral vectors (AAV-VEGF) stimulates skeletal muscle regeneration and enhances muscle function in mdx mice

19.20    Marco Sandri, Padua, Italy: Master genes of muscle atrophy and hypertrophy

 

Openings

 

Ugo Carraro

Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy

E-mail: ugo.carraro@unipd.it

 

Welcome address

It is my pleasure to welcome old and new friends and students at the PADUA MUSCLE DAYS 2006, which will provide room for fruitful discussions on muscle topics relevant to healing and rehabilitation of muscle diseases. This afternoon we will have three lectures from Professor Thordur Helgason, Reykjavik, Iceland on “Segmentation and modeling of thigh muscles, bones, veins and arteries”, Professor Giuseppe Vita, Messina, Italy on “VEGF gene transfer using adeno-associated viral vectors (AAV-VEGF) stimulates skeletal muscle regeneration and enhances muscle function in mdx mice” and Doctor Marco Sandri, Padua, Italy on “Master genes of muscle atrophy and hypertrophy”. They will provide an overview of the multidisciplinary approaches and results needed, if we all have the ambition to perform basic research with perspectives of clinical relevance. Then this evening we will have a light dinner to slip well and be ready for a very long Saturday. Tomorrow morning after the introductory words of Professor Vita, Scientific Coordinator of the PRIN 2004-2006 research program funded by COFIN-MIUR, Dr. Helmut Kern, Wien, Austria will open the session with a lecture on  “Functional electrical stimulation of denervated muscle: Clinical improvements”. I have no space to acknowledge all PRIN MEETING speakers, but let me remember that Professor Winfried Mayr, coordinator of the EU Program RISE (Use of electrical stimulation to restore standing in paraplegics with long-term denervated degenerated muscles) will lecture on “Denervated muscles in humans: currently used equipment for functional electrical stimulation and functional monitoring”. Then cell biologists, neurophysiologists, pathologists, bioengineers, and plastic surgeons report on recovery, repair, regeneration, healing and reconstruction of skeletal muscle. At the end of the PRIN MEETING we will have some time for General Discussion & Future Directions, in particular to evaluate our chances to be supported by the incoming 7° EU Program and by other national and international Grant Agencies. In the late afternoon, we will end these two PADUA MUSCLE DAYS with a series of short presentations from junior or senior researchers. Several of them work in the Virtual Laboratory of the Interdepartmental Research Center of Myology (cirMYO) of the University of Padua. You will find that very interesting results will be presented during this late afternoon session, e.g., Professor Scelsi, Pavia, Italy will encourage those interested to rescue muscle and motility of paraplegics with good news from his presentation “Flaccid paraplegia: Improvement of capillary supply after long term FES in human permanent lower motoneuron denervation”. Lastly, just for long viewers, I will present experimental evidence that endovascular electrostimulation of large skeletal muscles might be a clinical option, if nanotechnologists will spread into the capillary network soft electrodes to touch individual myofibers. In the future, this may provide artificial synapses to denervated muscles and tuned control to groups of innervated muscles. Results presented today by Icelander friends are cornerstones to build up these options.

I hope you will be gratified for your time in Montegrotto Terme (hopefully not all spent in the relaxing warm swimming pools) and for the formal and informal discussions with friends and students during and in between the Meeting Sessions.

Let we start with the forewords of Professor Francesco Mazzoleni and Professor Paolo Bernardi.

 

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Francesco Mazzoleni

Unit of Plastic Surgery, Department of Medical&Surgical Specialities, Interdepartmental Research Center of Myology, University of  Padua, Italy

E-mail: francesco.mazzoleni@unipd.it

 

Forewords

Let me first thank Professor Carraro for his kind invitation to the Padua Muscle Days 2006. He did most of the organizing efforts. When we started our collaboration some years ago, he, as a muscle biologist, contributed his basic knowledge, I, as a Surgeon, shared my experience on muscle traumathology and muscle grafting in reconstructive surgery. Both we were aware of the many scientific and technical gaps. Both us were motivated by the hope to finally provide some clinically relevant information. Fruitful informal discussion produced ideas and research programs. We were in full agreement on a couple of points: 1. Clinical research was urgently in need of broader and deeper knowledge on muscle regeneration potentials and constrains; 2. Biological approaches and constructs are also needed to attain muscle reconstruction. During the following years, much important scientific information had been provided by the international community. In the last years the stem cell field, the development of promising bioengineered materials, the explosion of molecular genetics and of the genetic engineering seemed to promise that our goals could be at hands. Tissue engineering based on in vitro construction of tissue and organs provides some clinical applications. The concepts of Regenerative Medicine and of the Guided Healing extended to the surgical approaches. All together, this advancement is changing minds and practices on the healing process and how to redirect it from the natural evolution of scarring toward the more useful process of regeneration. To be part of this culture, and each of us contributing what is dictated by his professional role, has been very stimulating and effective in the programming, execution and critical evaluation of our research activities and results. Surely we did our best with enthusiasm and passion. Produced our work relevant fruits? I couldn’t and I wouldn’t evaluate it alone. What I can say is that the collaboration changed some of my personal opinions and that it significantly changed my views and approaches on “the problem of the muscle managements in reparative and reconstructive surgery”. As usual, the list of the open queries is much longer than the list of accepted answers. The “muscle problem” needs a lot more research and even more discussions. The Meeting provides a forum: it is timely and relevant. Once again, thanks Professor Carraro!

 

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Session One

 

Thordur Helgason and Paolo Gargiulo

Landspitali, University Hospital, Reykjavik, Iceland

E-mail: thordur@landspitali.is

 

Segmentation and modelling of thigh muscles, bones, veins and arteries

In the frame of the European project RISE spiral CT scans are taken from three paraplegic patients in a regular interval of four months.  They have denervated and degenerated muscles in the lower extremities.  The scans deliver full three-dimensional description of the thigh tissue in the area from above the hip joint to down below the knee joint.  In this work the CT scans are segmented and each tissue type displayed to monitor changes in muscles, bone, veins and arteries in a period of electrical stimulation therapy of the thigh muscles.  Computer and calk models are made for investigation of the changes.  Results indicate that the method is adequate for monitoring work and its sensitivity enables detection of small changes in muscles and bone mineral density.

 

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Giuseppe Vita and Sonia Messina

Department of Neuroscience, Psychiatry and Anesthesiology, University of Messina, Italy

E-mail: giuseppe.vita@unime.it

 

Vascular endothelial growth factor gene transfer using adeno-associated viral vectors (AAV-VEGF) stimulates skeletal muscle regeneration and enhances muscle function in mdx mice

Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis. Several studies support its role in promoting muscle cells growth, differentiation and survival. We tested VEGF effect on muscle function, morphological and biochemical parameters in mdx and normal mice. One month after injection, AAV-VEGF treated muscles showed augmented expression of VEGF mean and VEGFR-2, the receptor mediating VEGF effect on muscle cell. VEGF treated mdx mice showed higher forelimb strength mean as well as higher strength normalized to weight. At quantitative histological evaluation of muscle, VEGF-treated mdx mice showed a reduction of necrotic fibers area and an increase of small centrally nucleated fibers area, marker of regeneration. In mdx mice, VEGF-treatment increased the number of cells positive for markers of early and late regeneration and also augmented the capillary density in regenerating fibers area. We report the novel observation of a beneficial effect of AAV-VEGF in mdx mice likely exerted by pro-regenerative and angiogenic effects.

 

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Marco Sandri
Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine & Department of Biomedical Science, University of Padua, Italy

E-mail: marco.sandri@unipd.it

 

Master genes in muscle hypertrophy and atrophy

The plasticity of skeletal muscle, namely the ability to change the size of its constituent fibers (muscle hypertrophy or atrophy) or their type (switching from a fast-glycolytic to a slow-oxidative phenotype or vice versa), is mainly dictated by use or disuse, namely by the amount and pattern of muscle activity that results from i) motor neuron firing patterns and ii) mechanical loading conditions. Changes in muscle fiber size and type affect muscle functional properties, including i) the amount of force generated during contraction, ii) the speed of shortening, that reflects myosin isoform composition, and iii) the resistance to fatigue, that depends by the mitochondrial oxidative enzymes. We will discuss here the signaling pathways controlling activity-dependent protein turnover in skeletal muscle fibers and will especially focus on the kinase Akt, also called protein kinase B (PKB) that has emerged as a major evolutionarily conserved signaling pathway in the control of cell size.

 

Saturday, October 21, 2006

 

Session Two

 

Meeting of the PRIN 2004-2006 Program: “Dalla rigenerazione alla ricostruzione del muscolo scheletrico: Meccanismi regolatori e applicazioni terapeutiche”

Skeletal Muscle: From Regeneration To Reconstruction

 

09.00           Giuseppe Vita and Ugo Carraro: Introduction

 

09.15    Helmut Kern, Wien, Austria: Functional electrical stimulation of denervated muscle: Clinical improvements.

10.00           Winfried Mayr, Wien, Austria: Denervated muscles in humans: currently used equipment for functional electrical stimulation and functional monitoring

10.20           Feliciano Protasi, Chieti, Italy: Electron microscopy of the human denervated muscle: Severe muscle atrophy and degeneration in spinal cord injury patients can be reversed by functional electrical stimulation (FES).

10.40           Reginald Bittner, Wien, Austria: Biochemical findings in electrically stimulated human long-term denervated muscle.

 

11.00    Break

 

11.20    Fabio Francini, Firenze, Italy: Effects of long-term denervation on excitation contraction coupling in single skeletal muscle fibers of the rat

11.40    Marina Marini, Bologna, Italy: Long-term variations in myogenic gene expression in denervated rat muscle

12.00    Ugo Carraro, Padova, Italy: Regeneration of the long-term denervated human muscle

 

13.00 Lunch

 

14.00           Giuseppe Vita, Messina, Italy: Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in mdx mice

14.30    Maria Cristina Tanzi, Milano, Italy: Scaffolds for muscle tissue engineering

15.00    Maria Rosa Melone, Napoli, Italy: A comparative analysis of different biomaterials in the engineering of skeletal muscle using C2C12 cells in vitro

15.30           Vincenzo Vindigni, Padova, Italy: A rat model for reconstruction of ablated muscle

16.00           General Discussion & Future Directions (7° EU Program and other Applications)

 

 

Giuseppe Vita, Scientific Coordinator of a PRIN 2004 research program funded by COFIN-MIUR

Department of Neuroscience, Psychiatry and Anesthesiology, University of Messina, Italy

E-mail: giuseppe.vita@unime.it

 

From regeneration to reconstruction of skeletal muscle: regulatory mechanisms and therapeutic applications

   There has been much debate on the capacity of skeletal muscle to undergo effective regeneration in patients with muscle disease secondary to trauma, myotoxic agents, ischemia/reperfusion, muscular dystrophies and other genetic myopathies, acquired dystrophic processes (e.g., long-term denervation or chronic disuse), etc.

   The project had four main aims: 1) To detail the biochemical and molecular events, modulated by activation of NF-kB pathway, underlying muscle necrosis and regeneration, and to study the effects of pharmacological blockade of NF-kB on muscle histopathology and function in the animal model of Duchenne muscular dystrophy (DMD). 2) To engineer muscle-like constructs from human muscle-derived stem cells by using polymer scaffolds, and to optimize the use of growth factors or bioactive molecules for improvement of the therapeutical potentialities; 3) To develop a methodology for reconstruction of ablated muscles and repopulation of long-term denervated muscles; 4) To study the therapeutical feasibility of electrical stimulation of reconstructed muscle in experimental chronic denervation.

   The project involved four units with different but complementary expertises: in particular, two units were coordinated by neurologists with specific interest in neuromuscular diseases, one unit by a bioengineer author of several patents on polymers for pharmaceutical and biomedical application, one unit by a physiopatologist with great experience in the study of muscle trophism restoring in different muscular diseases. The study project included possible therapeutic implications in DMD and chronic muscle denervation, and the cooperation with private companies highly specialized in polymer micro extrusion with experience also in biodegradable polymers for biomedical applications.

 

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Helmut Kern

Institut für Physikalische Medizin und Rehabilitation, Wilheminenspital Wien, Austria

E-mail: helmut.kern@wienkav.at

 

By using an adapted stimulation protocol with increased number of stimuli per day, thus increasing the amount of the muscle activity, we were able to improve the condition of the patient’s thigh muscles.  The perfusion was increased by 100% - 480% and muscle fiber size increased by +53%. This is also confirmed by measuring the muscle cross sectional area with computed tomography (CT) of the thigh showing an increase in m. quadriceps area of 34.6%. The electrically induced contraction force was improved by 828% in patients which were paralyzed up to 2 years. An additional effect of this therapy was the better cosmetical appearance of the thigh which is highly appreciated by the patients. Overall the above mentioned factors are an important contribution for preventing secondary diseases like decubital ulcers in patients with permanent/chronic denervation of the lower extremity.

 

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Winfried Mayr

Center of Biomedical Engineering and Physics, Medical University of Vienna, Austria

E-mail: winfried.mayr@meduniwien.ac.at

 

Denervated muscles in humans: currently used equipment for functional electrical stimulation and functional monitoring

Non-invasive functional activation of denervated muscles and assessment of muscle function require novel stimulation and measurement techniques. Stimulation equipment must be safe enough for home-based training though the applied electrical parameters raise critical safety issues. Assessment of biomechanical and electrophysiological parameters is provided by oscillation tonometry, twitch and m-wave measurements.

 

*****

 

Feliciano Protasi

Laboratory of Cellular Physiology, Interuniversity Institute of Myology , CeSI Centro Scienze dell'Invecchiamento, Università Gabriele d'Annunzio, Chieti, Italy

E-mail: fprotasi@phobos.unich.it

 

Electron microscopy of the human denervated muscle: Severe muscle atrophy and degeneration in spinal cord injury patients can be reversed by functional electrical stimulation (FES).

In spinal cord injury patients, Functional Electrical Stimulation induces recovery of muscle fibers at ultra-structural level (myofibrilles and excitation-contraction coupling apparatus) in complete absence of nerve endings. These studies, demonstrating that denervated muscle can be rescued by FES, are of main importance for the rehabilitation of paraplegics.

 

*****

 

 

 

 

Reginald E. Bittner
Center of Anatomy and Cell Biology, University of Vienna, Austria

E-mail: reginald.bittner@meduniwien.ac.at

 

Biochemical findings in electrically stimulated human long-term denervated muscle

The EU-funded RISE-project aims to better understand the effect of long term denervation on skeletal muscle and attempts to restore muscle tissue by functional electrical stimulation (FES) in paraplegic individuals. Muscle biopsies from human paraplegic patients (vastus lateralis) and from a pig animal model prior and after electrical stimulation show different stages of neurogenic atrophy and, thereafter irreversible dystrophy characterised by fatty and fibrotic tissue. The number and function of mitochondria progressively decline during the denervation in both humans and pigs, hereby establishing a new biomarker for functional impairments of denervated muscle. Application of electrical stimulation regimes can restore long term denervated skeletal muscles in human paraplegic patients to a degree which enables these patients to “RISE” from their wheel chairs standing up and walking along a bar. Despite the improvements, the mitochondrial function could not be fully restored, resulting in a limited muscle fatigue resistance in both humans and animals. Our findings should help to design more appropriate therapeutic FES regimens to functionally restore long term denervated muscle in paraplegia.

 

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Fabio Francini

Department of Physiological Sciences, University of Florence, Italy

E-mail: fabio.francini@unifi.it

 

Effects of long-term denervation on excitation contraction coupling in single skeletal muscle fibers of the rat

By electrophysiological methods we investigated in Soleus muscle the changes of the L-type Ca2+ current (ICa) and of the morphological features (fiber diameters, D; sarcolemnic, Cm, and tubular CT, surface area) induced by long-term denervation. The excitation-contraction coupling damage involved a depressed ICa and reduced values of D, Cm and CT. Notably, the effect of denervation was drastically more evident in the decrease of tubular than of sarcolemnic surface area. These effects were just evident after 3 weeks but the strongest damage was observed after 20 weeks of denervation. Moreover, also after 44 weeks skeletal fibers expressed affected but functional L-type Ca2+ channels.

 

*****

 

Marina Marini

Department of Histology, Embryology and Applied Biology, University of Bologna, Italy

E-mail: marina.marini@unibo.it

 

Long-term variations in myogenic gene expression in denervated rat muscle

In long-term denervation genes indexes of regenerative myogenesis or tissue remodelling are up-regulated, while genes indexes of muscle activity are down-regulated. Genes indexes of oxidative stress are also up-regulated in short-, medium-, or long-term denervated rat muscle. These and other data suggest that denervation induces oxidative stress. On the other hand, other indexes related to muscle activity, such as angiogenetic factors (VEGF, KDR) and energy usage indexes (AMPK, GLUT4), are down-regulated. Altogether, these data show the presence of an unexpectedly high adaptation activity in muscle tissue long after the loss of nerve stimulation. Taking into account the different lifespan of rats and humans, such results suggest that in humans the opportunity for therapeutic intervention may not have disappeared even years after the pathological event that led to denervation.

 

*****

 

Ugo Carraro

Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy

E-mail: ugo.carraro@unipd.it

 

Regeneration of the long-term denervated human muscle

Light and electron microscopy and antibody for embryonic myosin show that myogenic regenerative events are present in human muscle up to 37-year post-spinal cord injury. After 5 years of Functional Electrical Stimulation (FES) in the recovered muscles regenerative events are present, but at a lower rate than in long-term denervated muscles. We conclude that the FES-training of paraplegic subjects is safe (it does not induce more muscle damage/regeneration than denervation per se) and effective (the mean size of the myofibers went from 15.4 to 27.0, that is, a 76% increase after two years of FES).

 

*****

 

Giuseppe Vita

Department of Neuroscience, Psychiatry and Anesthesiology, University of Messina, Italy

E-mail: giuseppe.vita@unime.it

 

Nuclear factor kappa-b blockade reduces skeletal muscle degeneration and enhances muscle function in mdx mice

Nuclear factor-kB (NF-kB) is a transcription factor regulating the expression of genes involved in inflammatory, immune and acute stress responses of the cell. Several lines of evidence suggest a role of NF-kB in muscle degeneration and regeneration in Duchenne muscular dystrophy (DMD) patients and its animal model, the mdx mice. We investigated the effects of NF-kB blocking by IRFI 042 and PDTC on functional, biochemical and morphological parameters in mdx mice.

NF-kB inhibitors treated mdx mice showed: 1) an amelioration in functional parameters with an increased forelimb strength and strength normalized to weight, and decreased fatigue; 2) a reduction of muscle necrosis and an enhancement of regeneration. IRFI 042 blunted NF-kB DNA-binding activity and TNF-α expression in the dystrophic muscles. Moreover, IRFI 042 treated mdx mice had reduced serum creatine kinase levels, decremented markers of oxidative stress.

Our data suggest that oxidative stress/lipid peroxidation represents one of the mechanisms activating NF-kB and the consequent pathogenetic cascade in mdx muscles and that the inhibition of this cascade has beneficial effects. Further studies are needed to better elucidate the mechanisms of action of these drugs and therefore their possible therapeutic implications in DMD. 

 

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Maria Cristina Tanzi
BioMatLab, Department of Bioengineering, Politecnico di Milano, Italy

E-mail: mariacristina.tanzi@polimi.it

 

Scaffolds for muscle tissue engineering

Two different approaches can be followed in muscle tissue engineering: implantation of in vitro cultured polymer scaffolds, specifically designed to promote cellular orientation (e.g. micropatterned surfaces), or in vivo delivering of cells to the site of action by use of biodegradable carriers (e.g. microcapsules). Both strategies are currently developed at BioMatLab.

 

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Maria Rosa Melone

Department of Neurological Sciences, II University of Naples, School of Medicine, Naples, Italy

E-mail:  marina.melone@unina2.it

 

A comparative analysis of different biomaterials in the engineering of skeletal muscle using C2C12 cells in vitro

Modulation of tissue regeneration subsequent to injury or degenerative disease, by cell transplantation requires the survival of donor cells and their stable incorporation into the host tissue. Transplantation of cells on scaffolds, which activates the cells, promotes their outward migration and prevents premature terminal differentiation, combines the advantages of tissue regeneration obtained with direct cell injection with the control over transplanted cell fate made possible with the use of cell-instructive scaffolds. The last few years have marked a substantial paradigm shift in design criteria for modern synthetic biomaterials, fully integrating principles from cell and molecular biology: materials equipped with molecular cues mimicking certain aspects of structure or function of natural extracellular matrices (ECMs) and ECM-bound growth factors are quickly being developed. Muscle tissue engineering can be obtained by either in vivo or in vitro approach. The aims are to regenerate the myofibers using autologous cells and biodegradable materials (i.e. scaffold or carrier) as main components. The effective repair and regeneration of injured tissues and organs depend on early reestablishment of the blood metabolic support. As research at the interface between materials and biology increasingly overlaps, we look forward to seeing how each continues to inspire developments in the other.

 

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Vincenzo Vindigni

Plastic Surgery Clinic, Interdepartmental Research Center of Myology, University of Padua, Italy E-mail: vincenzo.vindigni@unipd.it

 

A rat model for reconstruction of ablated muscle

The goal of this study was to apply a step-by-step approach to identify factors favoring the survival of autologous satellite cells and, thus, muscle regeneration (“guided healing”). Muscle regeneration in free grafts is also determined by the size of the muscle being transplanted, because the speed at which new blood vessels penetrate into the mass of an avascular necrotic tissue will determine the extent of fibrosis. Auto-grafting of the middle third of the rectus abdominis muscle results in scar. In a rat model of full-thickness rectus abdominis muscle ablation, when autologous myoblasts were isolated from the explanted rectus abdominis and seeded in a homologous acellular matrix, the only regenerated myofibers are confined to the border of the patch. We succeed in regenerating myofibers in the graft by injecting marcaine in both the auto-graft and the surrounding muscles. Three weeks after surgery, the patch was paved by young centrally nucleated myofibers intermixed to early myofibers and myotubes, both expressing embryonic myosin. Muscle regeneration seems to be the result of co-ordinate migration of angioblasts and satellite cell–derived myoblasts from the muscles surrounding the patch, suggesting that (neo)vascularization of the scaffold followed by co-ordinate proliferation of seeded cells are mandatory events to allow myoblasts to migrate into the patch and differentiate up to myofiber stage.

 

 

Saturday, October 21, 2006

 

Session Three

 

Workshop of the Interdepartmental Research Center of Myology (cir-MYO)

 

17.00    Biral D, Rossini K, Caccavale S,  Adami N, Carraro U: Reinnervation events in long-term denervated rat muscles

17.15           Sferrazza R, Bassetto F, Mazzoleni F, Rossini K, Carraro U: Free neurovascular muscle flap: Long-term biopsy findings

17.30    Salviati A, Vindigni V, Bassetto F, Mazzoleni F, Biral D, Adami N, Carraro U: Myogenic events in muscle massive crushing

17.45    Rossini K, Biral D, Carraro U, Mayr W, Kern H: N-CAM and isomyosins of long-term denervated human muscle

18.00    Kern H, Boncompagni S, Protasi F, Rossini K, Biral D, Caccavale S, Adami N, Carraro U: Clinical and histological aspects of the thigh muscle in short-term and long-term upper motoneuron-complete spinal cord injury

18.15    Scelsi R, Poggi P, Carraro U, Mayr W, Kern H: Flaccid paraplegia: Improvement of capillary supply after long term FES in human permanent lower motoneuron denervation.

18.30           Borsato C, Dal Borgo R, Stramare R, Fanin M, Pegoraro E, Angelini C: Different muscle MRI imaging in polymyositis and dermatomyositis

18.45           Carraro U, Salviati A, Vindigni V, Mazzoleni F, Rossini K, Adami N, Kern H: Endovascular electrostimulation of rat muscles

 

 

Biral Donatella, Rossini Katia, Caccavale Susy, Adami Nicoletta, Carraro Ugo

Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padova, Italy

E-mail: ugo.carraro@unipd.it

 

Reinnervation events in long-term denervated rat muscles

Following permanent sciatectomy, the distal-leg muscles undergo a severe atrophy during the first 4-6 months of denervation, which ends in tissue degeneration after additional 3-5 months. After 1 month of denervation the myofiber diameter decreases to 50% of normal fibers. Progressively it reaches values of less than 20% during additional 2 months. Then a steady-state severe atrophy characterizes the surviving myofibers, whose number begins to decrease from 6 months. At 9-11 months of permanent denervation the normal fascicular architecture of the muscle is lost and interstitial tissue, which includes adipocytes and sheets of collagen, dramatically increases at the expenses of the myofibers. The ATPases demonstrate expression of a fast-like type of myosin, suggesting that fiber type differentiation is lost. In particular slow myosin disappears and only a fast-like myosin is present in the surviving fibers. Histochemical SDH, a marker of mitochondria of oxidative myofibers, decreases and finally disappears in both slow and fast myofibers. On the other hand, some regenerative myogenetic events randomly appear as demonstrated by anti-MHCemb monoclonals. Myotubes are also seen around atrophic fibers in very atrophic muscles. N-CAM, an adhesion molecule restricted to the synapse in innervated myofibers, is expressed along all atrophic fibers early after denervation. It also marks myotubes and early regenerated myofibers. It seems to be lost in the very atrophic myofibers at 9-11 months of denervation. In spite of the surgical approaches utilized to prevent reinnervation from the sciatic proximal stump, some reinnervation of the distal leg muscles occurs, resulting in minimal or substantial recovery of muscle mass and function. These reinnervation events are recognizable at the light microscopy level by size and morphology of the reinnervated myofibers and by the recovery of the fascicle muscle structure. A clear type grouping (clustering of muscle fibers with the same contractile and metabolic profile) is the golden standard of muscle reinnervation. The fascicles are made of well packed fibers positive for either fast or slow ATPase. Mitochondrial activity is demonstrated by succinic acid deidrogenase staining and vessels have normal distribution. While in normal soleus muscles a low percentage of type II fibers are sparsely distributed among type I fibers, in some area of the sciatectomized-reinnervated muscles type II and Type I fibers are grouped. The innervated fibers neither express embryonic myosin nor NCAM (outside the synapse). Indeed in muscle the levels of the neural cell adhesion molecule N-CAM are regulated in parallel with the susceptibility of muscle to innervation. In fact, it is expressed on the surface of early embryonic myotubes (until the nerve endings contact the myofibers), declines in level as development proceeds (i. e., it is restricted to the synaptic region), reappears when adult muscles are denervated and it is lost after reinnervation. So the lack of the expression of N-CAM confirms the state of innervation of the type-grouped fibers. How long rat or human myofibers re-express N-CAM during permanent denervation remains to be analyzed in details.

 

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Sferrazza Rossella, Vindigni Vincenzo, Bassetto Franco, Mazzoleni Francesco, Rossini Katia (1) Carraro Ugo (1)

Unit of Plastic Surgery and (1) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy –

E-mail: francesco.mazzoleni@unipd.it

 

Free neurovascular muscle flap: long-term biopsy findings

Muscle trophism depends on a variety of factors, among which the most important are vascularization, innervation and use. Therefore, a chronically denervated muscle is subjected to a transformation: first of all it becomes atrophied, with a decrease in muscle fiber diameter and number. Then it becomes dystrophic: myofibers degenerate and are substituted by fatty and connective tissue. In plastic surgery free neurovascular flap is used with reconstructive purposes. In spite of the great number of improvements, aesthetic and functional results of free flap are not always successful, mostly because fibrosis and fatty change take place after some years (muscle lipodystrophy). Otherwise, in a few number of cases the transplanted muscle (graft) is reinnervated by axons that are found in the grafting area. Spontaneous reinnervation lets the muscle preserve a certain degree of trophism. Apart from the muscle’s possible transformation, in free flap myofiber regeneration events occur starting from activated satellite cells. These are myogenic precursors which are able to become myoblasts when muscle is damaged for different reasons. In this study, six biopsies were taken from six different free neurovascular flaps made in a period of six years (1997-2003). The average age of subjects (5 male, 1 female) was 36 years, average time to biopsy was 4 years and 5 months, range: 3 - 7 years). Biopsies were studied histologically and immunohistochemically to determine degree of muscle trophism, embryonic myosin, fast and slow myosin, N-CAM proteins. Biopsies were analyzed morfometrically and myofiber, connective and fatty tissue distribution determined. The results confirm that long time denervated muscle tissue becomes atrophied in different degrees. In some cases activation of satellite cells is evident and several years after the neurovascular free flap transfer the muscle still attempts to regenerate. Spontaneous reinnervation was also observed. Pro- and anti-reinnervation factors will be discussed.

 

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Salviati Alessandro, Vindigni Vincenzo, Bassetto Franco, Mazzoleni Francesco, Biral Donatella (1), Adami Nicoletta (1), Caccavale Susy (1), Rossini Katia (1), Carraro Ugo (1)

Unit of Plastic Surgery and (1) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy

E-mail: salviati.alessandro@unipd.it

 

Myogenic events in muscle massive crushing

The events of muscle degeneration and regeneration were studied in an experimental model of mechanical and myotoxic injury in rats. Rectangular patches, 24mm long and 8mm wide, of rat rectus abdominis muscle were processed by mechanical crushing and injection of marcaine. Three days after the operation a second injection of marcaine was performed. The patches, with a small amount of surrounding abdominal wall, were harvested respectively at 11 and 24 days from the operation. In a second group of animals, 3 days after the crushing-marcaine operation, the patch was used as a muscle autograft, and harvests were performed at 24 days. Sections of the biopsies harvested at 11 days showed wide destruction of muscle tissue, infiltration by inflammatory cells, and wide presence of small regenerating myofibers positive to an anti-embryonic myosin monoclonal antibody (anti MHC-emb). On the other hand, sections of the 24 days harvests showed an almost complete reconstruction of the muscle tissue in the patch, with well packed large myofibers negative to anti MHC-emb, but distinct ATPase staining (fats and slow regenerated and reinnervated myofibers). Sections of the autografted muscle harvested at 24 days showed a dominant fibrotic component, with a few regenerating myofibers still positive to anti MHC-emb.

The results suggest that even after massive mechanical-myotoxic damage, conservation of some continuity of the vascular network and of the connective tissue architecture with the surrounding tissue maintain the potential of muscle tissue to regenerate and to be reinnervated.  Conversely when total interruption of the vascular and nervous network is performed, regenerative events are confined to the rim and the main result is a fibrous scar tissue of the patch.

 

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Rossini Katia, Biral Donatella, Carraro Ugo, Mayr Winfried (1), Kern Helmut (2)

Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy; (1) Center for Biomedical Engineering and Physics, Medical University of Vienna, Austria; (2) Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Wilhelminenspital, Vienna, Austria

katia.rossini@unipd.it

 

N-CAM and isomyosins of long-term denervated human muscle

In rodents 1-year of permanent denervation ends in lipodystrophy of the muscle tissue. Indeed the normal fascicular architecture of the muscle is lost and interstitial tissue, which includes adipocytes and sheets of collagen, dramatically increases at the expenses of the myofibers. The ATPases of the residual myofibers demonstrate expression of a fast-like type of myosin, suggesting that fiber type differentiation is lost. In particular slow myosin disappears and only a fast-like myosin is present in the surviving fibers. Histochemical SDH, a marker of mitochondria of oxidative myofibers, decreases and finally disappears in both slow and fast myofibers.

The biopsy bank of the European Project RISE (contract n. n. QLG5-CT-2001-02191) provides for the first time the chance to study long term effects of permanent denervation on human muscle tissue after spinal cord injury (SCI). Main results at light microscopy level are: 1. Human skeletal muscle tissue undergoes three phases during long-term lower motor neuron denervation: i) Atrophy (up to 18 months post-injury); ii) Lipodystrophy (3 – 10 year post-injury); iii) Fibrosis (after 10-year post-injury). The three phases change over time at a much slower rate than in rodents (years vs. months); 2. Repeated cycles of myofiber death/regeneration contribute to long-term trophism of human muscle tissue after permanent lower-motoneuron denervation.

Histochemical and immunohistochemical analyses show that the checkerboard appearance of the human denervated tissue is maintained at least up to 2 year post-SCI, but generally a fast-like ATPase staining prevails. At longer time the severely atrophic myofibers show if any, a very light staining. Small or large sparse regenerating myofibers, that is, those positive to an anti-embryonic myosin monoclonal antibody (anti MHC-emb) are present in almost all the biopsies. About 1% of the myofibers present in the cryosections are anti-MHCemb positive. These regenerated myofibers are also stained with an antibody recognizing N-CAM, an adhesion molecule restricted to the synapse in innervated myofibers, but expressed along all atrophic fibers early after denervation in rodents and humans. It also marks myotubes and early myofibers of rat muscles after myotoxic injury. Since during aneural myogenesis transition from MHCemb to a fast-like isoform(s) occurs, while N-CAM restriction to synapse is inhibited, we believe that myofiber regeneration accounts for much more of the long term denervated human myofibers than now accepted. On the other hand, beside in the regenerated myofibers, the expression of N-CAM seems to be lost in the atrophic myofibers of the long-term SCI subjects in both large and small size myofibers, suggesting that the re-expression of this molecule is possibly restricted to the early periods of SCI. Anyhow, comparing pre- and post-functional electrical stimulation (FES) an induced increase in the mean size of the myofibers (from 15.4 to 27.0, i.e., 76% increase at two-year FES) occurred in the series of RISE subjects.

 

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Kern Helmut, Boncompagni Simona (1), Protasi Feliciano (1), Rossini Katia (2), Biral Donatella (2),  Caccavale Susy (2), Adami Nicoletta (2), Carraro Ugo (2)

Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Wilhelminenspital, Vienna, Austria; (1) IIM - Interuniversitary Institute of Myology, CeSI - Center for Research on Ageing, University G. d’Annunzio, Chieti, Italy; (2) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padova, Italy

E-mail: helmut.kern@wienkav.at

 

Clinical and histological aspects of the thigh muscle in short-term and long-term upper motoneuron-complete spinal cord injury

Subjects with complete lesion of the spinal cord at the level of T3/4 – T11 develop a so called spastic paraplegia. In these patients muscles of the lower limb are still innervated by the motor neurons that, however, are anymore controlled by the central nervous system.

The aim of this study was to determine the progression of muscle atrophy, from functional, histological and ultrastructural point of view in upper motor neuron paretic patients. For this purpose we have compared one group of short term paralyzed paraplegic patients (6 patients, ten biopsies, 2.2 ± 0.5, 1.6 to 3.0 years from injury) to one group of long term paralyzed paraplegic patients (4 patients, 8 biopsies, 17.0 ± 2.6, 14.0 to 20.3 years from injury).

The subjects of the short term group developed a torque of 26.33 ± 15.21 Nm and the subjects of the long term group 16.64 ± 7.8 Nm respectively during stimulation induced isometric knee extension. Mean m. quadriceps cross sectional area and tissue density, measured 20 cm proximal to the knee joint, was 49.84 ± 12.76 cm² with mean density of 41.37 ± 4.39 HU in the short term paralyzed patients and 38.97 ± 5.09 cm² with mean density of 38.89 ± 2.37 HU in the long term paralyzed subjects.

The percentage of tissue types within the muscle biopsies of short-term paralyzed subjects was: muscle fibers: 70.5 ± 11.8 %; fatty tissues: 3.6 ± SD 3.8 %; collagen: 1.8 ± 2.9 %; other connective tissue: 24.1 ± 8.3 %.   The distribution of tissue types in long-term paralyzed thigh muscles was: muscle fibers: 72.8 ± 4.8 %; fatty tissues: 5.8 ± SD 4.8 %; collagen: 2.8 ± 3.0 %; other connective tissue: 18.7 ± 7.8 %. Muscle fiber diameters was 34.4 ± 9.9 µm (mean+/-SD) in the short-term and 37.4 ± 7.5 µm in the long term groups. Fiber size spectra suggest that this small increase in the long-term group is the result of a decreased percentage of severely atrophic myofibers (41 % in short versus 23% in the long term patients). The comparison of the two groups of patients indicates no significant changes from either functional or histological point of view between short and long term paralyzed muscle groups. Size distribution and ultrastructural characteristics of some myofibers suggest that minor lower motoneuron denervation is present in both groups and contributes to the functional outcomes of the thigh muscles. On the other hand, electron microscopy shows that even after long periods of paralysis, muscle fibers maintain a quite organized contractile apparatus, with distinct myofibrils and sarcomeres, while the major alteration appear to be a misplacement of the activating and metabolic machinery. These findings indicates that in spastic patients the initial rapid loss of muscle mass occurring in the first months after the injury is followed by a prolonged steady state in which muscle maintains its remaining mass, composition, and performance for extended periods of time, i.e. up to 20 years. The maintenance of muscle is likely the result of spontaneous reflex activity, since we have previously shown that in muscles that loose their peripheral motor innervation progression of atrophy is much more severe and finally ends in tissue degeneration.

All together, our results confirm that there are not upper-time limits to start recovery of the mass and function of paretic muscle by body weight-supported treadmill training (BWSTT) and/or Functional Electrical Stimulation (FES).

 

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Scelsi Roberto, Poggi Paola (1), Carraro Ugo (2), Kern Helmut (3)

Department of Human Pathology and (1) Department of Human Anatomy, University of Pavia, Italy; (2) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padova, Italy; (3) Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Wilhelminenspital, Vienna, Austria

E-mail: "apat" <apat@unipv.it>

 

Flaccid paraplegia: improvement of the muscle capillary supply after early-started daily functional electric stimulation (FES) in human permanent lower motoneuron denervation

Force and endurance of leg muscle of spastic paraplegic patients (i.e., with lesion of the upper motoneuron) are suboptimal even after successful rehabilitation strategies such as body weight-supported treadmill training (BWSTT), electrically induced cycle training (EICT)  and Functional Electrical Stimulation (FES).  Even more difficult is to normalize muscle performance when the injury involves lower motoneurons (permanent flaccid paraplegia) since many months after spinal cord injury (SCI) muscle atrophy is complicated by fibrosis and fat substitution. We are testing the hypothesis that capillary network changes and /or muscle blood perfusion control may contribute to the unsatisfactory muscle performance. Here we report histological and ultrastructural analyses of capillary supply in permanent motoneuron-denervated human skeletal muscles. Capillary intramuscular network was studied in two groups of patients enrolled in the RISE Trial, without or after a new life-long FES training. Vastus lateralis biopsies from four patients with traumatic spinal cord and conus-cauda lesions of 9 months – 3 years (group 1) were compared to biopsies from two patients with similar lesions since 9.6 and 10.6 years, who underwent 7.7 and 9.3 years of FES training, respectively (group 2). Spinal motoneuron-denervated patients show muscle fiber atrophy and degeneration with fat substitution. Mean number of capillaries per fiber was reduced (2.0 +/- 0.1 SE) in comparison to normal adult muscle (4.10 +/- 0.2 SE). Numerous small vessels show endothelial changes and thickening and/or duplication of basal lamina. In the two subjects, who underwent several years of daily FES training, the vascular network appears almost normal in number (mean number of capillaries per fiber: 3.4 and 3.6, respectively) and ultrastructural morphology. Meantime light and electron microscopy analyses show an evident recovery of myofiber size and structure with significant reduction of fatty infiltration. We believe that these preliminary observations strongly support FES as a strategy to recover mass and function of long term denervated muscle, in particular when the FES regime starts early after SCI.

 

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Borsato Carlo, Dal Borgo Roberto (1), Stramare Roberto (1), Fanin Marina, Pegoraro Elena, Angelini Corrado

Department of Neurosciences, and (1) Department of Radiology University of Padova, Italy

E-mail: carlo.borsato@unipd.it

 

Different muscle MRI imaging in polymyositis and dermatomyositis

To determine whether muscle MRI imaging could reveal different radiological patterns in patients affected with polymyositis and dermatomyositis. Our study included 7 patients (5 females, 2 males; mean age 52 years) affected with polymyositis and 2 patients (1 female and 1 male; mean age 23 years) affected with dermatomyositis. All patients received a diagnosis of polymyositis or dermatomyositis on the base of clinical, laboratory and histopathological features. Seven patients received diagnosis of polymyositis and two patients received diagnosis of dermatomyositis. A muscle MRI study (T1, STIR sequences) was performed. The fibro-fatty changes have been evaluated on T1 sequences. Myoedema has been evaluated on STIR sequences. Both polymyositis and dermatomyositis patients presented progressive proximal muscle weakness, myalgia and high creatine kinase levels. Muscle biopsy showed different specific histopathological patterns: necrotic fibers invaded by leucocytes in patients affected with polymyositis, perifascicular atrophy and leucocyte invasion in patients affected with dermatomyositis. Muscle MRI study revealed characteristic patterns: in T1 sequences we observed in both groups of patients minimal fibro-fatty changes, involving particularly the posterior compartments of lower limbs; in STIR sequences we observed a diffuse signal hyperintensity due to muscle inflammation in all districts with particular involvement of anterior compartments of proximal lower limbs. However, while in patients affected with polymyositis we observed a strong, diffuse intra-fascicular oedema and a slight inter-fascicular oedema with no subcutaneous involvement, in patients affected with dermatomyositis we observed a different pattern with strong inter-fascicular oedema with slight intra-fascicular oedema. In this group of patients a subcutaneous inflammatory involvement was, also, observed. Polymyositis and dermatomyositis represent the two most common autoimmune inflammatory myopathies. Both diseases could show similar clinical features, with progressive proximal muscle weakness and myalgia and it could be difficult distinguish the two forms of myositis, particularly if there are minimal cutaneous manifestations. Histopathological aspects on muscle biopsy are important because the two forms of myositis might be characterized due to their different pathogenetic mechanisms. In dermatomyositis, there is activation of complement that leads to the formation and deposition of membranolytic attack complex on the endomysial microvasculature, resulting in capillary necrosis, microinfarcts, endofascicular hypoperfusion, and finally perifascicular atrophy. In polymyositis there is evidence of an antigen-directed and MHC-I-restricted cytotoxicity mediated by CD8 T cells. Muscle MRI study in both groups of patients revealed different pattern of muscle inflammation distribution, particularly in lower limbs. In STIR sequences we observed a diffuse signal hyperintensity with different distribution: in polymyositis we observed a more severe intra-fascicular oedema with slight inter-fascicular and no subcutaneous involvement while in dermatomyositis we observed a distinctive radiological aspect with a strong involvement of inter-fascicular and subcutaneous spaces. These radiological differences could be explained by different pathogenetic mechanisms involving the two forms of inflammatory myopathies which cause, also, different oedema distribution.

 

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Carraro Ugo, Rossini Katia, Adami Nicoletta, Salviati Alessandro (1), Vindigni Vincenzo (1), Mazzoleni Francesco (1), Kern Helmut (2)

Laboratory of Applied Myology of the Department of Biomedical Science and (1) Unit of Plastic Surgery, Interdepartmental Research Center of Myology, University of  Padua, Italy and (2) Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Wilhelminenspital, Vienna, Austria

E-mail: ugo.carraro@unipd.it

 

Endovascular electrostimulation of rat muscles

Functional electrical stimulation of skeletal muscle is performed by cutaneous surface electrodes, transcutaneous needle electrodes or epineural implanted electrodes. From several months after injury, denervated muscle can only be electrostimulated by large transcutaneous surface electrodes or subcutaneous perimisial electrodes.

We are exploring the hypothesis that customized endovascular electrodes either via arteries or veins may be inserted in the denervated muscle up to the capillary tree. In spite of the success of the endocardial electrostimulation for cardiac pacing, risks of this approach are related to the: i) lower excitability of the denervated muscle in comparison to innervated tissue or myocardium; ii)  thrombogenesis and embolism, iii) traumatic muscle damage related to muscle contraction.

Some of these risks ask for new materials and technologies (possibly related to nanotechnology approaches) and therefore of very new ideas and methods. On the other hand, we will show that besides standing on sound biological bases, preliminary acute studies in a rat model demonstrate the feasibility of the new concept. The plasticity of the muscle tissue and of its vascular net may be the basis to further test our dream.

 

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Abstracts of the Invited Speakers

 

Bittner Reginald E.
Center of Anatomy and Cell Biology, University of Vienna, Austria

E-mail: reginald.bittner@meduniwien.ac.at

 

Biochemical findings in long term denervated and FES muscles in humans and pigs

Palsy and atrophy of skeletal muscles in paraplegic patients not only leads to physical inabilities but also to pressure ulcers due to the missing muscle- cushion. The EU-funded RISE-project aims to better understand the effect of long term denervation on skeletal muscle and attempts to restore muscle tissue by functional electrical stimulation (FES) in paraplegic individuals. We investigated muscle biopsies from human paraplegic patients (vastus lateralis) and from a pig animal model (experimental denervation of the tibialis anterior and the extensor digitorum logus muscle) prior and after electrical stimulation. We examined morphological and biochemical parameters with special emphasis on the mitochondrial enzymes to gain insight in the energy metabolism of the diseased muscles. We found that the skeletal muscle shows different stages of neurogenic atrophy and thereafter enters irreversible dystrophic stages characterised replacement of muscle tissue by fatty and fibrotic tissue. The number and function of mitochondria progressively declined during the denervation period of in both, humans and pigs, hereby establishing a new biomarker for functional impairments of denervated muscle. We show that application of electrical stimulation regimes can restore long term denervated skeletal muscles in human paraplegic patients to a degree which enables these patients to “RISE” from their wheel chairs standing up and walking along a bar. In addition, no pressure sores occurred in FES-treated paraplegic patients, indicating a protective effect of FES to prevent these frequent and threatening complications. Despite morphological and functional improvements achieved by the electrical stimulation regimens, the mitochondrial function could not be restored in the same extent resulting in a limited fatigue resistance in electrically stimulated paraplegic muscles in humans and animals. Our findings should help to design more appropriate therapeutic FES.regimens to functionally restore long term denervated muscle in paraplegics.

 

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Francini Fabio, Roberta Squecco, Helmut Kern (1), Feliciano Protasi (2), Donatella Biral (3)l, Katia Rossini (3), Ugo Carraro (3)

Department of  Physiological Sciences, University of Florence, Italy; (1)Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Wilhelminenspital, Vienna, Austria; (2) Laboratory of Cellular Physiology Cesi, Center for Research on Ageing, Fondazione Università Gabriele d'Annunzio Chieti, Italy; (3) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy

E-mail: fabio.francini@unifi.it

 

Effects of long-term denervation on excitation-contraction coupling in single skeletal muscle fibers of the rat

By electrophysiological methods we investigated in Soleus muscle the changes of the L-type Ca2+ current (ICa) and of the morphological features (fiber diameters, D; sarcolemnic, Cm, and tubular CT, surface area) induced by long-term denervation. The excitation-contraction coupling damage involved a depressed ICa and reduced values of D, Cm and CT. Notably, the effect of denervation was drastically more evident in the decrease of tubular than of sarcolemnic surface area. These effects were just evident after 3 weeks but the strongest damage was observed after 20 weeks of denervation. Moreover, also after 44 weeks skeletal fibers expressed affected but functional L-type Ca2+ channels.

 

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Abruzzo Prevalenza, Rossini Katia (1), CarraroUgo (1), Biral Donatella (1), Kern Helmut (2), Lapalombella Rosa, Di Tullio Simona, Biondi Annalisa (3), Lenaz Giorgio (3), Marini Marina

Department of Histology, Embryology, and Applied Biology, University of Bologna, Italy; (1) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padova, Italy; (2) Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Vienna, Austria; (3) Department of Biochemistry, University of Bologna, Italy.

 

Oxidative stress and gene expression variations in denervated rat muscle

By using Real Time Quantitative PCR, five selected genes, indexes of stress and/or oxidative stress (HSP70, HSP27, SOD-1, SOD-2, GSTM1), were found to be upregulated in short- (15 days), medium- (3 months), or long- (9 months) term denervated rat Tibialis Anterior. SOD activity was evaluated by enzymatic methods and a remarkable correspondence was found with gene expression. A concurrent increase in reactivity with the fluorescent probe DCF confirmed that denervation induces oxidative stress. However, the amount of antioxidant defenses decrease while ROS apparently increase with time from denervation. Whose events are the primary cause of such an oxidative stress is still an unresolved question, since no sign of inflammation is evident, at least in medium- and long-term denervated muscles, and mitochondrial morphology is apparently preserved. In any case, this finding suggests that antioxidant supplementation may be helpful for denervated patients.

 

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Lapalombella Rosa, Rossini Katia (1), Carraro Ugo (1), Biral Donatella (1), Kern Helmut (2), Alessandra Scordari, Marini Marina

Department of Histology, Embryology, and Applied Biology, University of Bologna, Italy; (1) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padova, Italy; (2) Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Department of Physical Medicine, Vienna, Austria

 

Gene expression variations in medium- and long-term denervated rat muscle

Expression of 11 selected genes was studied by Real Time Quantitative PCR in medium- (3 month) or long- (9 month) term denervated rat skeletal muscle. Data show that genes whose up-regulation is index of regenerative myogenesis (Myogenin, MyoD, Mrf4, Myh3) or tissue remodelling (Cals2, Mmp2) are still up-regulated 9 months after denervation. Immuno-histochemistry with the antibody directed against the embryonic isoform of heavy-chain myosin suggests that such changes in gene expression occur as focal events among degenerated myofibers.

On the other hand, other indexes related to muscle activity, such as angiogenetic factors (VEGF, KDR) and energy usage indexes (AMPK, GLUT4), are down-regulated. Altogether, these data show the presence of an unexpectedly high adaptation activity in muscle tissue long after the loss of nerve stimulation. Taking into account the different lifespan of rats and humans, such a result suggests that in humans the opportunity for therapeutic intervention may not have disappeared even years after the pathological event that led to denervation.

 

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Melone Mariarosa Anna Beatrice, Lus Giacomo, Petillo Orsolina (1), Calarco Anna (1), Torpedo Angela (1), D’Apolito Maria (1), Tanzi Maria Cristina (2), Faré Silvia (2), Draghi Lorenza (2), Peluso Gianfranco (1)

Department of Neurological Sciences-Second University of Naples, Italy; (1) Institute of Biochemistry of Proteins-CNR, Naples, Italy; BioMatLab, Bioengineering Department, Politecnico di Milano, Italy

 

A comparative analysis of different biomaterials in the engineering of skeletal muscle using C2C12 cells in vitro

Tissue engineering represents a possible approach to replace the lost or defective muscle. The purpose of this study was to assess a new scaffold design for muscle tissue engineering, by comparing the growth of C2C12 mouse cells on different polymers. Polymeric substrata were prepared with a soft lithography technique (replica molding) from three different masters (a milled aluminium plate, a laser-processed alumina sheet, and a microgroove modeling paste mould obtained from a home-made fibers array). Each master imprinted with an elastomeric material (Elastosil® RT601 Wacker), was in turn used as a mould to prepare the microgrooved polymeric substrata by solvent-casting. Polymer selected was a medical grade biodegradable poly(L-lactic acid) /trimethylencarbonate (PLLA/TMC 68:32, Boerhinger Ingelheim). Four different groups based on fiber spacing (30 to 35, 50 to 55, 70 to 75, and 90 to 95 µm) were evaluated. We compared 3-week growth of C2C12 cells cultured on scaffolds alone, or supplementing the scaffold with two key cytokines involved in muscle regeneration, bFGF (30±120 pM) and HGF (70±280pM). Both methods facilitated cell attachment, growth, and viability. The cells lined the inner and outer surfaces of the scaffold, filling the pores, as demonstrated by scanning electron microscopy and histology. Immunohistochemical staining of BrdUrd incorporation confirmed high levels of cellular proliferation. Yet, the combination of both cytokines showed a strong synergistic stimulation of C2C12 myoblast proliferation and chemotactic activity. After 21 days in the HGF-treated samples large areas of well-formed and aligned myotube fascicles were present which appeared to be more frequent than in the scaffold-alone samples and to show higher expression of differentiation-associated proteins. Our results show that the combination of PLLA/TMC polymer as a vehicle for myoblasts and exogenous HGF as a proliferative stimulator is a good candidate for the generation of skeletal muscle in vitro.

 

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Vindigni Vincenzo, Mazzoleni Francesco, Salviati Alessandro, Rossini Katia (1), Carraro Ugo (1)

Unit of Plastic Surgery and (1) Laboratory of Applied Myology of the Department of Biomedical Science, Interdepartmental Research Center of Myology, University of Padua, Italy

E-mail: vincenzo.vindigni@unipd.it

 

A rat model for reconstruction of ablated muscle

Studies in mice, rats, and humans show that the efficiency of muscle regeneration in grafts is determined in part by the thickness of the muscle being transplanted, because the speed at which new blood vessels penetrate into the mass of avascular necrotic tissue will determine the extent of fibrosis. The goal of this study was to apply a step-by-step approach to identify factors favoring the survival of autologous satellite cells and, thus, muscle regeneration (“guided healing”). In a rat model of full-thickness rectus abdominis muscle ablation, autologous myoblasts were isolated from the explanted rectus abdominis and seeded in a homologous acellular matrix immediately after wall reconstruction (group 1, five animals). In group 2 (five animals), the ablated rectus abdominis was autografted in situ. In a third group of ten rats, marcaine is injected in the autograft (5 animals) or acellular muscle matrix (5 animals), and into the surrounding abdominal wall muscle at the time of surgery. Histological (haematoxylin-eosin stain) and immunofluorescence (antibody to embryonic myosin) analysis were performed three weeks after surgery. Patches have the mechanical properties to support the abdominal organs (survival rate 100%, hernia rate 0%). Auto-grafting of the middle third of the rectus abdominis muscle results in scar. When autologous myoblasts are seeded in a homologous acellular matrix, the only regenerated myofibers are confined to the border of the patch. We succeed in regenerating myofibers in the graft by injecting marcaine in both the auto-graft and the surrounding muscles. Three weeks after surgery, the patch was paved by young centrally nucleated myofibers intermixed to early myofibers and myotubes, which express embryonic myosin. Muscle regeneration seems to be the result of co-ordinate migration of angioblasts and satellite cell–derived myoblasts from the muscles surrounding the patch. The results strongly suggest that vascularization of the scaffold followed by co-ordinate proliferation of the seeded cells are mandatory events to allow myoblasts to migrate into the patch and differentiate up to myofiber stage.

 

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Vita Giuseppe, Sonia Messina

Department of Neuroscience, Psychiatry and Anesthesiology, University of Messina, Italy

 

Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in mdx mice

Nuclear factor-kB (NF-kB) is an ubiquitous transcription factor regulating the expression of a plethora of genes involved in inflammatory, immune and acute stress responses. Several lines of evidence suggest a role of NF-kB in muscle degeneration and regeneration in Duchenne muscular dystrophy (DMD) patients and mdx mice. We investigated the effects of NF-kB blocking by IRFI 042 and PDTC on functional, biochemical and morphological parameters in mdx mice. IRFI 042 is a synthetic vitamin E analogue with a strong inhibitory activity on both oxidative stress/lipid peroxidation and NF-kB activation. PDTC is a synthetic antioxidant able to prevent NF-kB inhibitor (IkB-a) degradation. NF-kB inhibitors treated mdx mice showed: 1) an amelioration in functional parameters with an increased forelimb strength (p<0.05) and strength normalized to weight (p<0.05), and decreased fatigue with PDTC (p<0.05); 2) a reduction of muscle necrosis (p<0.01) and an enhancement of regeneration (p<0.05). We found that IRFI 042 blunted NF-kB DNA-binding activity and TNF-α expression in the dystrophic muscles (p<0.01). Moreover, IRFI 042 treated mdx mice had reduced serum CK levels (p<0.01), decremented muscle conjugated dienes content and augmented muscle reduced glutathione (p<0.01). Our data suggest that oxidative stress/lipid peroxidation represents one of the mechanisms activating NF-kB and the consequent pathogenetic cascade in mdx muscles and that the inhibition of this cascade has beneficial effects in mdx mice. Further studies are needed to better elucidate the mechanisms of action of these drugs and therefore their possible therapeutic implications in DMD. 

Messina S, Bitto A, Aguennouz M, Minutoli L, Monici MC, Altavilla D, Squadrito F, Vita G. Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice. Exp Neurol 2006;198:234-41.

Messina S, Altavilla D, Aguennouz M, Seminara P, Minutoli L, Monici MC, Bitto A, Mazzeo A, Marini H, Squadrito F, Vita G. Lipid peroxidation inhibition blunts nuclear factor-kappa B activation, reduces skeletal muscle degeneration, and enhances muscle function in mdx mice. Am J Pathol 2006;168:918-26.

 

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Vita Giuseppe, Sonia Messina

Department of Neuroscience, Psychiatry and Anesthesiology, University of Messina, Italy

 

Vascular endothelial growth factor gene transfer using adeno-associated viral vectors (AAV-VEGF) stimulates skeletal muscle regeneration and enhances muscle function in mdx mice

Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis. Several studies support its role in myogenesis and in myoblast migration and survival. Recently it has been reported that the delivery of VEGF using adeno-associated-virus (AAV) vectors reduces muscle damage and promotes muscle regeneration in experimental models of muscle necrosis.  We tested VEGF effects on muscle function, histopathology, immuno-histochemistry and biochemical parameters in mdx and wild type mice. We performed intramuscular administration of AAV-VEGF or AAV-LacZ in biceps and tibialis anterior muscles. One month after injection, AAV-VEGF treated muscles showed augmented expression of VEGF (mean p<0.05) and VEGFR-2 (mean p<0.01) compared to controls. VEGF treated mdx mice showed higher forelimb strength (mean +19.5%, p<0.05) as well as higher strength normalized to weight (+ 14.9; mean%, p<0.05). At quantitative morphological evaluation, VEGF-treated muscles showed a reduction of necrotic fibers area (p<0.05) and an increase of small centrally nucleated fibers area (p<0.05). VEGF-treated mdx mice muscles exhibited an increased number of cells positive for markers of early and late regeneration, respectively myogenin and developmental myosin heavy chain (p<0.05), and also an augmented capillary density in regenerating fibers area (p<0.05). We report the novel observation of a beneficial effect of AAV-VEGF in mdx mice exerted mainly by pro-regenerative and angiogenic effects. Further studies are needed to better clarify the mechanisms of action of VEGF and therefore the possible therapeutic implications in Duchenne muscular dystrophy. 

S. Messina, M. Aguennouz, A. Bitto, A. Migliorato, M. Giacca, F. Squadrito, G. Vita

Vascular endothelial growth factor gene transfer using adeno-associated viral vectors stimulates skeletal muscle regeneration and enhances muscle function in mdx mice (abstract). Neuromuscular Disorders 2006;16:624.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Scientific Secretariat: Prof. Ugo Carraro Department of Biomedical Science Viale G. Colombo, 3 – 35121 – Padova – Italy Tel: +39 049 8276030; Fax +39 049 8276040 e-mail: ugo.carraro@unipd.it

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