the coil placed on L3-L5 beside the midline initially for lumbosacral stimulation, which was variable to obtain the
maximal movement of their hindlimbs as an optimal stimulation (Fig 1).
Figure 1: The site of stimulation
This
stimulation of the rats performed for 60min/day, for 10 days. Magnetic stimulation for
the rats of the stimulated group was started 1 day after the operation. The day
after the stimulation period ended, the soleus, tibialis anterior and extensor digitorum longus muscles of the rats were
surgically removed from both legs, immersed in RNA later TM (TaKaRa
BIOCHEMICALS) twelve hours.
The muscle samples were pulverized under liquid
nitrogen and homogenized in cold ISOGEN (NIPPON GENE) (1.0ml). After homogenisation,
proteins and insoluble material were removed by 10min of centrifugation at 4 deg
C at 12.000g. Phase separation was performed using chloroform (0.25ml). Isopropanol (0.25ml) were used for RNA precipitation.
Pellets were resuspended in 70% ethanol (1.0ml). RNA
concentration was assessed spectrophotometrically.
1.0 μg of Total RNA were reverse-transcripbed using Avian Myoblastosis Virus reverse Transcriptase XL (Life Science) 0.25U/μl and Random 9mers 2.5μM in 30second, at 42 deg C. Polymerase
Chain Reaction (PCR) were
performed using Takara TaqTM polymerase (TAKARA) and the primers
specific to MHC
IIb, MHC IId(x), MHC IIa, and MHC Iβ (Table 1) which were derived from published cDNA sequences. Depending on the initial amount of template, the number of cycles was determined to 28 cycles to allow product detection in the exponential range of amplification. PCR products were analyzed by agarose gel electrophoresis, aliquots (10.0μl) were loaded in a 1.5% agarose gel containing 0.5μg/ml ethisium bromide and separated in a 1X TBE buffer. Band intensity and area were calculated using NIH image. In order to investigate changes in transcript level of selected genes in MHC, we performed a semi-quantitative analysis of the message level of MHC genes. Each band intensity was calculated relative to the GAPDH control and expressed.
3
Results
Soleus. The
mean relative amount of expression in each mRNA of MHC IIb, MHC IId(x),
MHC IIa, and MHC Iβ to GAPDH were, respectively, 1.19, 1.12, 0.89
and 0.713 in control group, 0.563, 0.293,0.679 and 0.317 in non-stimulated
group, 1.183, 1.51, 1.23 and 0.579 in stimulated group. The emerging rate of MHC Iβ and MHC IIa was remarkably changed. The emerging of
non-stimulated groups was decreased and that of stimulated group was affected
to increase.
Tibialis anterior. The mean relative amount of expression in each
mRNA of MHC to GAPDH were, respectively, 0.438, 0.702, 0.782 and 0.337 in control
group, 0.559, 0.677, 0.566 and 0.152 in non-stimulated group, 0.578, 0.883,
0.625 and 0.476 in stimulated group. For the TA we detected no differences
among the experimental groups.
Extensor digitorum longus.
The mean
relative amount of expression were, respectively, 0.648, 0.647, 0.665 and 0.348
in control group, 0.503, 0.591, 0.560 and 0.074 in non-stimulated group, 0.610,
0.450, 0.467 and 0.198 in stimulated group (Fig. 2). In the EDL, a decreasing
in mRNA for non-stimulated group, and furthermore, only the increasing of
emerging of MHCIβwas quantified. It would be said that the data suggests the transition to slow
muscle may occurred. 
Figure 2: Distribution of MHC mRNA isoform in Soleus Muscle
4 Discussion and Conclusions
The present
study was undertaken to investigate, in rat fast-twitch muscle exposed to
stimulation, transitions in MHC expression at the mRNA level. Fast twitch muscles
can be transformed into slower contracting muscles by chronic low-frequency
stimulation in order of MHC Iβ → MHC IIa → MHC IId(x) → MHC IIb [4]. This process encompasses fast-to-slow fiber-type transitions concomitant
with sequential changes in MHC composition. We were especially interested in
the temporal patterns of induced transition in various MHC mRNA isoforms. Because sequences of the three fast MHC isoforms and MHC Iβ are available.
The muscle atrophy in patients with SCI has been demonstrated a progressive decrease in the fiber diameter and changes in the fiber type distribution [4]. Once the muscle atrophy has occurred before the stimulation started and developed, then it require a longer period of time till the muscles return to near normal condition. In order to restore paralyzed muscles by TES, an increase in muscle fiber diameter is required. It is important to maintain muscle power by TES, as well as an increase in the size of muscle fiber. Our preliminary experiment suggests that magnetic stimulation of peripheral nerve might affect to prevent slow-to-fast transition. As a result of using magnetic stimulation, slow type of muscle growth in the acute phase of hindlimb suspension might outpaces fast muscle atrophy. We believe that SCI patients can receive FMS earlier as long as the muscle atrophy does not develop.
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