carbon dioxide ( co2 ) water bathing has a long history in european countries and 
 has been used to treat cardiovascular disease since the middle ages . co2 water 
 bathing is a kind of remedy with a wide spectrum of applications . co2 water 
 bathing results in vasodilatation and an increase in blood flow1 , 2 . the vasodilatation 
 is induced by co2 , which diffuses into the subcutaneous tissue through the skin . for example , co2 water bathing enhanced vegf mrna levels , resulting in 
 neovascularization in ischemic hindlimbs of mice3 . in our previous study satellite 
 cells are muscle stem cells and play an important role in skeletal muscle fiber repair after 
 injury . satellite cells proliferate , differentiate into myoblasts expressing muscle - specific 
 proteins , fuse into myotubes , and finally mature into myofibers5 . satellite cells are normally in a quiescent state , but they 
 are activated when skeletal muscle fibers are injured . the activated satellite cells 
 proliferate , differentiate , and fuse to repair injured skeletal muscle fibers . myogenic 
 regulatory factors ( mrfs ) , including myod , myf5 , myogenin , and mrf4 , are transcription 
 factors that control the expression of several muscle genes . the myogenic basic 
 helix - loop - helix ( bhlh ) family of transcription factors are thought to play an important 
 role in the development and regeneration of skeletal muscle6 , and mrfs regulate the complex phenomenon of myogenic 
 differentiation . as noted above , mrfs play an important role in skeletal muscle regeneration , but myod and 
 myogenin were not increased by co2 water bathing at 2 weeks after injury in our 
 previous study4 . therefore , the mechanism 
 underlying the acceleration of skeletal muscle regeneration induced by co2 water 
 bathing was unclear . the expression of mrfs is observed in the early stage of regeneration 
 after skeletal muscle injury . we hypothesized that co2 water bathing increases 
 the expression of myod and myogenin in early stage of regeneration after injury , since the 
 expression of myod and myogenin proteins was observed in a morphological study at 4 days 
 after skeletal muscle injury7 . thus , the 
 purpose of this study was to examine the effect of co2 water bathing on the 
 expression of myod and myogenin in injured muscles at 4 days after skeletal muscle 
 injury . the rats were housed in a 
 temperature controlled room , 22  c , on a 12 h:12  h light - dark cycle , and were allowed free 
 access to food and water . the rats were assigned to no - injury ( ni , n=4 ) , injury ( ic , n=4 ) , 
 injury + tap water bathing ( itw , n=4 ) , and injury + co2 water bathing 
 ( ico2 , n=4 ) groups . the rats in the ic , itw , and ico2 groups were 
 anesthetized by injection of pentobarbital sodium ( 50  mg / kg ) ; then , the left tibial anterior 
 ( ta ) muscle was injured by injection of 0.3  ml of 0.5% bupivacaine hydrochloride ( marcaine , 
 astrazeneca , osaka , japan ) using a disposable syringe with a 27-gauge needle . the needle was 
 inserted into the mid - belly portion of the ta muscle and advanced longitudinally to the 
 proximal portion . one day after the ta muscle 
 injury , rats in the itw , and ico2 groups were immersed in tap water and 
 co2 water ( co2 concentration ; 1,000  ppm ) , respectively , at 37  c , 
 for 30 minutes once a day for 3 consecutive days . co2 water containing a high 
 concentration of co2 was made from high pressure co2 in a cylinder and 
 tap water using an mre - spa ( mitsubishi rayon co. , ltd . , four days after 
 injury , the rats were sacrificed by injection of an overdose of sodium pentobarbital and 
 their left ta muscles were removed . the ta muscles were immediately frozen in isopentane 
 cooled by liquid nitrogen and stored at  80  c until analysis . all procedures were approved 
 by the animal care and use committee of kibi international university . for the analysis of the expression of myod and myogenin , the muscles were homogenized in 
 tris - hcl ( ph 7.4 ) . the protein concentrations of these samples were measured using the bradford method 
 and a coomassie protein assay kit ( thermo fisher scientific k.k . , the 
 samples were added to ezapply ( atto , tokyo , japan ) , adjusted to a final protein 
 concentration of 1  g/l , and then boiled at 95  c for 5 minutes . we then applied 10  g 
 protein to a 12.5% polyacrylamide gel ( atto , tokyo , japan ) . electrophoresis was carried out 
 at a constant current of 20 ma / gel for 60 minutes , and proteins were then transferred to a 
 polyvinylidene difluoride ( pvdf ) membrane using a horizblot 2  m ( atto , tokyo , japan ) at a 
 constant current of 2 ma / cm for 60 minutes . following blots , the pvdf membrane 
 was then incubated for 60 minutes using ezblot ( atto , tokyo , japan ) . the membrane was then 
 incubated with a monoclonal antibody for myod ( santa cruz biotechnology , santa cruz , ca , 
 usa ) diluted 1:2,000 with 0.1  m tris - hcl ( ph 7.5 ) or myogenin ( santa cruz biotechnology , 
 santa cruz , ca , usa ) diluted 1:2,000 with 0.1  m tris - hcl ( ph 7.5 ) for 1 hour at room 
 temperature . the membrane was washed 3 times in 0.1  m tris - hcl with 0.1% tween-20 and then 
 reacted with anti - mouse igg ( nacalai tesque , kyoto , japan ) diluted 1:10,000 with 0.1  m 
 tris - hcl ( ph 7.5 ) for 1 hour at room tempareture . the membrane was washed 3 times a further 
 in 0.1  m tris - hcl with 0.1% tween-20 and then reacted with ezwestblue ( atto , tokyo , japan ) . bands from western blots were quantified using the imagej software ( nih , bethesda , md , 
 usa ) . when there were 
 significant differences , the tukey post hoc test was used to determine differences between 
 the groups . statistical analyses were performed using excel statistics 2008 ( social survey 
 research information co. , ltd . values are means  sd , * ; different from ni , p < 0.01 . # ; different from ic , p < 
 0.05 .  ; different from itw , p < 0.05 our results are shown in table 1 . the myod 
 level was significantly higher in the ic , itw , and ico2 groups than in the ni 
 group ( p < 0.01 ) . however , there were no significant differences among the ic , itw , and 
 ico2 groups . the myogenin level was significantly higher in the ic , itw , and 
 ico2 groups than in the ni group ( p < 0.01 ) . in addition , the myogenin level 
 was significantly higher in the ico2 group than in the ni and itw groups ( p < 
 0.05 ) , but there was no significant difference in its level between the ic and itw 
 groups . in the present study , co2 water bathing increased the expression of myogenin , 
 but not the expression of myod . this result indicates that co2 water bathing 
 accelerates skeletal muscle regeneration via an increase in the expression of myogenin . myod and myogenin are transcription factors belonging to the bhlh family of transcription 
 factors . these proteins play important roles in satellite cell proliferation , 
 differentiation , fusion , and the formation of myocytes . myod has a critical role in skeletal 
 muscle regeneration via activation of satellite cells8 . cooper et al . observed that myod is expressed in myotubes with 
 centrally located myonuclei at 5 days after cardiotoxin injection9 . they also suggested that myod is required for proliferation 
 and differentiation of activated satellite cells during the regeneration of skeletal muscle 
 fibers . myotube formation is delayed in regenerating skeletal muscle in the absence of 
 myod10 , indicating that myod plays an 
 important role in myotube formation in regenerating skeletal muscle . in experiments using 
 knockout mice , myod and myf5 were shown to have roles in the determination of the myogenic 
 cell fate and the formation of myoblasts during embryogenesis , and myogenin and mrf4 were 
 shown to have roles in the activation of muscle differentiation11 , 12 . in addition , 
 newborn mice deficient for both myod and myf5 are devoid of myoblasts6 , and myogenin ( / ) mice form myoblasts that fail to form 
 myotubes13 . in the present study , activation of 
 satellite cells and myoblast formations occurred normally in the injured skeletal muscle 
 fibers in the groups with skeletal muscle injury , and the increase in myod was similar in 
 the ic , itw , and ico2 groups . these data suggest that co2 water 
 bathing did not affect the expression of myod , indicating that co2 water bathing 
 does not accelerate myoblast formation via activation of satellite cells . an increase in 
 myogenin was also observed in the groups with skeletal muscle injury . myotube formation 
 occurred normally in the groups with skeletal muscle injury ; however , the increase in 
 myogenin was greater in the ico2 group than in the ic and itw groups . this result 
 indicates that co2 water bathing affects the expression of myogenin , and might 
 accelerate fusion of myoblasts and formation of myotubes . in our previous study , co2 water bathing increased myonuclei in regenerating skeletal muscle fibers at 
 2 weeks after skeletal muscle injury . taken together , these results indicate that 
 co2 water bathing accelerates skeletal muscle fiber regeneration via increase 
 in the expression of myogenin enhancing the fusion of myoblasts . nitric oxide ( no ) is a key 
 signal responsible for satellite cell activation and hepatocyte growth factor ( hgf ) , which 
 is an activator of satellite cells , is released from the extracellular matrix after skeletal 
 muscle injury14 . tatsumi et al . reported 
 that activation of satellite cells and hgf release in stretched muscle were dependent on 
 local no production15 . reported that co2 water bathing increased in phosphorylated no synthase in 
 ischemic skeletal muscle3 , indicating that 
 co2 water bathing promotes no production in skeletal muscle . although no 
 measure of nos was included in the present study , we speculate that no production in 
 skeletal muscle was promoted by co2 water bathing further studies are needed to determine 
 more clearly the mechanisms which accelerate skeletal muscle fiber regeneration in 
 co2 water bathing .
[ purpose ] we reported that carbon dioxide ( co2 ) water bathing accelerates 
 skeletal muscle regeneration ; however , the underlying mechanism was unclear . 
 myod and 
 myogenin play roles in muscle regeneration , and the purpose of this study was to determine 
 changes in myod and myogenin caused by co2 water bathing after injury . 
 [ subjects ] sixteen female wistar rats 
 ( n = 4 per group ) were used . 
 [ methods ] the rats were 
 divided into four groups : no - injury ( ni ) , injury ( ic ) , injury + tap water bathing ( itw ) , 
 and injury + co2 water bathing ( ico2 ) . 
 muscle injury was induced by 
 injection of bupivacaine hydrochloride into the left tibial anterior ( ta ) muscles . 
 tap 
 water and co2 ( 1,000  ppm ) water bathing were performed at 37  c for 30 minutes 
 once a day . 
 the left ta muscles were removed 4 days after injury , and the expressions of 
 myod and myogenin were measured . 
 [ results ] myod and myogenin were increased in the ic , 
 itw , and ico2 groups compared with the ni group . 
 although the myod level was 
 similar in the ic , itw , and ico2 groups , myogenin increased more in the 
 ico2 group than in the ic and itw groups . 
 [ conclusion ] co2 water 
 bathing after muscle injury appears to induce an increase in the expression of 
 myogenin .