- 77 - The Regenerating Muscle as an Experimental Model for the Study of Factors which Affect Muscle Differentiation or Adaptation Menotti Midrio, Aram Megighian and Daniela Danieli-Betto Department of Human Anatomy and Physiology, University of Padova, Padova, Italy Abstract Bupivacaine-induced regeneration was studied in the rat soleus muscle in the presence or ab- sence of innervation, in the presence of tetrodotoxin (TTX)-induced block of nerve impulse conduction, and/or in the presence of vinblastine-induced block of nerve axoplasmic flow. Part of experiments were carried out on tenotomized muscles. Regenerated muscles were analysed for myosin heavy chain (MHC) composition 14 days after bupivacaine injection. In TTX-paralysed-regenerated muscles type 1 and type 2A MHC isoforms were not expressed. In denervated-regenerated muscles type 1 isoform was lacking, while all fast isoforms (2A, 2B, 2X) were expressed. Tenotomy alone increased type 2A fibres, but did not modify the ef- fects of surgical or functional denervation. Vinblastine-block caused up-regulation of 2A iso- form expression in non-tenotomized muscles. The results confirm the essential role played by neuromotor impulses for type 1 and type 2A isoform expression. They also support the hypothesis that axoplasmic flow carries some chemical factor inhibiting 2A isoform expres- sion. Key words: neurotrophic factors, regeneration, skeletal muscle, tenotomy, tetrototoxin, vinblastine. Basic Appl Myol 12 (2): 77-80, 2002 The muscle regenerating after an acute degeneration is an useful experimental model for studying factors which direct or affect muscle differentiation, and muscle plastic adaptation to functional demand. Regeneration starts from undifferentiated (satellite) cells, and through the myoblast and myotubes stages it ends with the adult, differentiated cells. Then, a sequence of events occurs which is similar to foetal myogenesis, with the advan- tage that the regenerating muscle allows greater experi- mental manipulations than the foetal muscle. Moreover, it is possible to affect the phenotype expression in the very initial steps of muscle differentiation. The interest of our laboratory is mainly focused on the role of innervation on muscle differentiation. Innervation can affect muscle properties by the pattern and/or the fre- quency of motor impulses and the release of chemical factors by nerve fibres (for references see [7]). In our in- vestigation, we tried to dissociate these two possible components of nerve influence by selectively blocking impulse conduction or axoplasmic flow in the nerve dur- ing muscle regeneration, and by comparing the effect of the blocks with that of surgical denervation of regenerat- ing muscle. Since it is known that the terminal phenotype of regenerating muscle is changed in the absence of nor- mal postural load [3], we also investigated the interac- tions between a particular kind of muscle postural un- loading, such as caused by tenotomy, and surgical or functional denervation on the pattern of regeneration. Materials and Methods All experiments were carried out according to the Helsinki Accords for Human Treatment of Animals during Experimentation. The study was approved by the Ethics Committee of the Medical Faculty of the University of Padova. All surgical procedures were performed under general ether anaesthesia. Acute degeneration was induced in the soleus (slow) muscle of the rat by injecting 0.5-0.7 ml of 0.5% bupivacaine solution [1] in the muscle ex- posed through a small cutaneous incision. Surgical de- nervation was performed by cutting the sciatic nerve near the trochanter. Chronic block of impulse conduc- tion was achieved by superfusion of sciatic nerve with tetrototoxin (TTX, Sankyo, Japan, 500 µg/ml), released from a miniosmotic pump (Alzet 2002, 200 μl volume, 0.5 μl/h release rate, 14 days discharge time)[2]. The pump was implanted in the interscapular region and connected by means of a polyethylene catheter to a sili- con cuff surrounding the nerve [5, 7]. The axoplasmic flow was blocked by applying around the nerve, for 20 min, a cotton wool soaked with 0.15 mM vinblastine