| Research Abstract |
INTRODUCTION
Free functioning muscle transplantation has often been employed to solve devastating problems of the extremities such as brachial plexus injury, Volkmann's contracture, and extensive muscle defects after trauma or oncological resection 1,2. However, the availability of a donor site is limited and final results of this transplantation depend on multiplefactors. Recently, repair and regeneration of adult skeletal muscle has been described 3. The satellite cells are normally mitotically quiescent. When stimulated by damage, some fractions of satellite cells are activated to reenter the cell cycle and/or to express myogenic regulatoryfactors (MRFs). The resulting myoblasts subsequently differentiate and fuse to form new replacement myofibers. Little is known, however, about the expression pattern of MRFs in the grafted muscle, even though experimental research of free muscle transplantation has been reportedby several authors. Our aim in this study was to search for evidence of
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the expression of MyoD, one of the MRFs, in an in vivo rat musclegrafting model.
METHODS
Experimental model for muscle transplantation and surgicalprocedures : 50 rats age 9 weeks old were used for autotransplantation of gastrocunemius muscles. 5 rats were used as a control. All muscle graftprocedures were performed aseptically under anesthesia with ketamine chloride and xylazine. The medial head of gastrocunemius muscle andits pedicle were exposed. After dissecting the proximal and distal bone insertions of the muscle, the whole body of the muscle was elevated as avascularized island muscle flap. The tibial artery was clamped for 1 hour to simulate a free graft. During this ischemic condition, three differentoperations were performed : the tibial nerve was cut and neurorrhaphy was performed (Reinnervation group, n=33), the tibial nerve was keptintact (Ischemic group, n=6), the tibial nerve was cut and not sutured (Dennervation group, n=6). The flap was returned to the muscle deficitand both ends were sutured to the tibial periosteum with 4-0 monofilament nylon sutures to return to the original muscle tension. Rats were allowed to move freely without external fixation postoperatively. On day 1,3 and 5,and week 1,2,3,4,5,6,7,and 8 after surgery, muscle specimens were harvested. Analysis of Electromyography (EMG) : EMG was performed for each group at 2,4 and 6 week after surgery, to evaluate reinnervation process. Analysis of Histology : Muscle specimens were fixed in 10% buffered formalin, embeddede in paraffin, sectioned and stained with hematoxylin-eosin (HE) at each harvest time. Semiquantitative RT-PCR : The muscles were pooled in the liquid nitrogen at each harvest time. RNA was extracted by the guanidium thiocyanate-phenol-chloroform extraction method 4. First strand cDNA was synthesized from 5 g of total RNA using T-Primed First-Strand Kit (Amersham Biosciences). RT-PCR was carried out using RT-PCR Beads (Amersham Biosciences) with primer sets, synthesized following the published sequence of the rat MyoD and GAPDH 5. Amplified products were electrophoresed using 2% agarose gel and stained with ethidium bromide. The gel was visualized under UV illumination and densitometric measurements were performed (Scion Image, Scion Corporation). The band density of PCR products was normalized to that of GAPDH.
RESULTS
Macroscopic findings : The transplants, when removed, showed a decrease in volume, compared to the time of grafting. The transplants older than 4 weeks twitched during dissection. EMG results : In the reinnervation group, low amplitude M-waves were observed on an evoked potential 4 weeks after transplantation (Figurel), and the muscle contracted with electrical stimulation. Histological results : At 2 and 4 weeks after neurorraphy, myoblasts were increased, satellite cells appeared, and muscle fibers were atrophic. At 6 weeks after surgery, cellularity decreased and muscle fibers were getting stronger. Expression of MyoD : The normal muscle had no expression of MyoD mRNA. In the reinnervation group, MyoD expression was observed at day 3 after surgery, peaked at week 4,and tapered off week 6 (Figure 2). In contrast, MyoD expression disappeared by 4 weeks post operation in the ischemic and denervation group (Figure 3). Comparison of relative ratio of MyoD/GAPDH at 3 weeks after transplant showed that the reinnervation group had larger MyoD expression than the denervation group.
DISCUSSION
The principle purpose of muscle transplantation is to restore motor functions and achieve activities of daily living. MyoD has an important role as a one of the regulators of regeneration of muscle. In this study, the expression of MyoD in the grafted muscle had a characteristic time course. This time course seems to be related to the reinnervation period, confirmed by EMG and histological analysis. Thus, it is suggested that MyoD has a critical role in the regeneration process of grafted muscle. The exact complex regulatory pathways remain poorly understood, however better understanding of the cellular and molecular process of transplanted muscle regeneration are important and may be beneficial for the development of therapeutic strategies for stable operative results and postoperative functions. Less
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