The Effect of Transcutaneous Application of Carbon Dioxide (CO2) on Skeletal Muscle
1
Oe, K; +1 Miwa, M; 2 Sakai, Y; 1 Niikura, T; 1 Lee, S Y; 1 Koh, A; 1 Koga, T; 1 Dogaki, Y; 1 Ueha, T; 1 Kurosaka, M
+1 Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
2
Faculty of Health Care Sciences, Himeji Dokkyo University, Himeji, Japan
[email protected]
INTRODUCTION
The presence of the carbonated spa (carbonated therapy) has been
known for a long time in the Europe, and it is known that carbonated
therapy is effective against cardiac disease and skin trouble. However
there has been no report investigating the effect of the carbonated
therapy on muscle.
We hypothesized that the transcutaneous application of CO2 to muscle
exhibited similar effect to the exercise training. In this study, we applied
CO2 to the rat lower limbs transcutaneously, and investigated the effect
on the fast muscle (tibialis anterior muscle; TA muscle). The changes of
the gene expression and protein in TA muscles were analyzed.
MATERIALS AND METHODS
Experimental set up
The use of animals was approved by the Animal Care and Use
Committee of Kobe University Graduate School of Medicine. 14 male
Sprague Dawley rats of 5 weeks of age were randomly divided into 2
groups (treatment group (CO2): transcutaneous CO2 application; non
treatment group (Control)). We used 100% CO2, and transcutaneous
CO2 absorption enhancing hydrogel (Hydrogel) and CO2 adaptor that
seals the body surface and retains the gas inside. Then, the area of skin
with Hydrogel was sealed up, and CO2 gas was flowed into the area.
(International patent application number: WO2004/002393)
Procedure for CO2 treatment
Under pentobarbital anesthesia (6 ml/kg), we cut the hairs of right
lower limbs, and apply Hydrogel to these lower limbs. The CO2 adaptor
was put on limbs, and CO2 was administrated into the adaptor for 10
minutes. This treatment was performed twice a week for 12 weeks.
Surgical Procedure
Rats were sacrificed after 12 weeks, and TA muscles of right lower
limb were dissected carefully and harvested.
Determination of the relative mitochondrial copy number
Genomic DNA was isolated from a 10 mg transversal slice of midbelly
TA using the GenElute Mammalian Genomic DNA Miniprep Kit.
Mitochondrial DNA (mtDNA) content relative to peroxisome
proliferators-activated receptor (PPAR)-gammma coactivator-1 (PGC1α) gene was measured using Real-time PCR as described previously.
[1].
Histchemical staining
Unfixed frozen TA muscles were sectioned at 10 µm thickness in the
cryostat and the ATPase staining was performed as previously described
[2]. The condition was pH 4.6.
Isolation of myosin heavy chain (MHC)
The protein solution was isolated from a 10 mg transversal slice of
midbelly TA using urea buffer. 5-20 µl of this homogenate were used for
MHC analysis by using two-dimensional electrophoresis. The MHC
analysis was as described previously [3]. After electrophoresis, gels
were silver-stained by using a Wako Silver Stain Kit. Images of MHC
bands were analyzed by LAS-3000, and the band intensities were
quantified by Scion-Image.
Statistics analysis
Data are shown as the mean values ± standard deviation. Results of two
groups were analyzed using "unpaired" Student's t tests. P < 0.05 was
taken to be statistically significant. (*, p < 0.05, **, p < 0.01)
RESULTS
TA Muscle Weight (Table 1)
The upward tendency of muscle weight and muscle weight/body
weight ratio in CO2 group was observed (n = 14).
Real-time PCR (Figure 1)
The gene expression of PGC-1α and Mitochondria in CO2 group was
significantly increased (n = 12).
MHC isoform (Figure 2)
The percentage of IIB fibers was decreased, and those of IID fibers
and/or IIA fibers in CO2 group were increased. The significant
difference in the percentages of IIB fibers and IIA fibers between two
groups was observed (n = 12).
ATPase Staining (Figure 3)
Figure 3 shows the deep layer of TA muscle. In this layer of CO2 group,
IIB fibers transformed to IIA. I fibers seemed unchanged.
Table 1
Control
CO2
Muscle weight(mg)
1.101 ± 0.190
1.112 ± 0.128
Muscle weight/body weight ratio
0.178 ± 0.023
0.188 ± 0.021
Figure 1
Figure 2
Control
CO2
Figure 3
White：IIA
Gray：IIB
Black：I
Control
CO2
DISCUSSION
Generally, the muscle fibers transform from IIB fibers to IID fibers and
IIA fibers in the aerobic exercise situation, whereas in long term bed rest
situation they transform to IIB fibers. The result of our study showed
that the muscle fibers in which CO2 was transcutaneously applied
transformed from IIB fibers to IID fibers and/or IIA fibers.
PGC-1α is known to be up-regulated under the aerobic exercise [4].
The muscle fiber transformation mechanism relates to the increase of the
mitochondrial quantity. PGC-1α increases the mitochondrial quantity [4].
Our results demonstrated that PGC-1α was up-regulated by
transcutaneous CO2 application, which correspondingly increased the
mitochondrial quantity. These data indicated that transcutaneous CO2
application to TA muscles under the non-exercise conditions may
possess similar effect to the aerobic exercise.
The current study suggests that the transcutaneous application of CO2
may have therapeutic potential for muscular strength recovery of disuse
atrophy in post operative patients and elderly people.
REFERENCE
[1] van den Broek NM et al, ASEB J. 2010;24:1354-64.
[2] Brooke MH, Kaiser KK. 1970;23:369-79.
[3] Saitoh A et al, J Appl Physiol. 1999;86:1483-9.
[4] Iwabu M et al, Nature 2010:29;464:1313-9.
Paper No. 431 • ORS 2011 Annual Meeting