a wound refers to a case where the continuity of a normal skin structure is destroyed by 
 physical damage to the skin , and the process of recovering such discontinuity is referred to 
 as wound healing1 . wound healing consists 
 of three phases  inflammation , proliferation , and remodeling  and wound healing progresses by 
 cells playing their unique roles in each phase2 . thus far , 13 types of collagens acting importantly in the wound healing process have been 
 discovered , and among them , type i accounts for 85% of the wound healing process3 . collagens engage in normal or pathological 
 bioprocesses such as cell combination , cell differentiation , wound healing , all kinds of 
 fibroses and inflammatory responses , and cancer transformation4 . collagen synthesis in fibroblasts is influenced by diverse cytokines including transforming 
 growth factor 1 ( tgf-1 ) , insulin - like growth factor 1 , interleukin 1 , and platelet - derived 
 growth factor , and in particular , tgf-1 is known to play a major in vivo role5 . tgf-1 , which is secreted from fibroblasts , 
 macrophages , and platelets , triggers chemotaxis of fibroblasts , macrophages , and monocytes . stimulation of fibroblasts increases synthesis and secretion of collagen fibers and other 
 substrate components and inhibits secretion of fiber decomposition enzymes . tgf-1 also 
 stimulates endothelial blood vessel cells , thereby promoting formation of blood vessels6 , 7 . kloth noted that electrical stimulation applied to wounded skin tissues increased 
 angiogenesis and perfusion , which raised the activity of granulation tissues and 
 fibroblasts , thereby promoting wound healing speed9 . physical therapy approaches to wound healing include hvpcs , 
 microcurrents , low - intensity laser , and ultrasound10,11,12,13 . hvpcs has a monophasic 
 twin peak pulse wave form and very short pulse duration and therefore can safely stimulate 
 deep tissues without damage14 . the 
 effects of hvpcs related to wound healing are a decrease in chronic decubitus ulcer , 
 inhibition of staphylococcus aureus growth , an increase in perfusion around ischemic wounds , 
 and improved microcirculation15,16,17,18 . doran et al . observed that hvpcs with the cathode as an 
 active electrode decreased the size of the inflammatory area19 . lee et al . applied hvpcs to the wound areas of rats for seven days 
 and observed that the experimental group s wound healing rate and fibroblast and collagen 
 density were significantly higher than those of the control group10 . jeong and song applied hvpcs to normal skin of rats for 
 seven days and noted that expression of collagen i and tgf-1 was promoted in the dermis 
 layer20 . however , most previous studies have concerned hvpcs with a non - visible contraction 
 intensity , and research on the effects of hvpcs with a visible contraction intensity on 
 wound healing is lacking . accordingly , the aim of this study was to compare the expressions 
 of tgf-1 and collagen i at each time point by applying hvpcs with a non - visible contraction 
 intensity to wound areas of white rats , thereby presenting a theoretical groundwork for its 
 clinical practice . thirty - six white adult male sprague - dawley rats ( ntacsam : sd , samtako bio korea , osan , 
 republic of korea ) , which were six weeks old and weighed from 200 to 220  g , were used and 
 were equally and randomly assigned to experimental group i , experimental group ii , and the 
 control group ( n = 12 per group ) . hvpcs with a non - visible contraction intensity was applied 
 to experimental group i , hvpcs with a visible contraction intensity was applied to 
 experimental group ii , and placebo stimulation was applied to the control group under the 
 same condition as used for the experimental groups after wounds were triggered . for 
 histological sections , four rats from each group were sacrificed on the third , fifth , and 
 seventh days . during the experimental period , whether the rats had any trauma or disease was 
 checked , and stress factors were minimized by reducing environmental changes . the 
 temperature and humidity of the breeding room were maintained at 22  2  c and 55  10% , 
 respectively . the light - dark cycle was set at 12 hours to maintain the constant conditions 
 in the breeding room during the experimental period . the rats were allowed to freely take 
 water and solid feed ( samyang co. , republic of korea ) . the institutional animal care and use 
 committee at dongshin university approved the protocols used in this study , and the animals 
 were cared for in accordance with the guidelines for animal experiments ( 2010 ) . the rats were subjected to general anesthesia using a respiratory anesthetic , sevoflurane , 
 and the hair on their back area was removed using a depilatory cream ( veet depilatory cream , 
 reckitt benckiser , france ) . after removing the hair , the skin was disinfected with 75% 
 alcohol so that no depilatory cream remained on the skin . a wound with a diameter of 10  mm 11 surgical blade at locations more than 
 15  mm laterally distant from the center . the panniculus carnosus was exposed to induce 
 wounds in a circular form , and the wound areas were dressed with sterilized gauze . the rats 
 rested for 24 hours after induction of the wounds without any manipulation in order to 
 minimize the stress and physiological response resulting from the wounds21 . the skin tissue of a rat has a panniculus 
 carnosus layer , and therefore induction of a circular wound only may induce a wound healing 
 speed similar to that of humans22 , 23 . the wounds were covered with sterilized gauze soaked with normal saline . an active 
 electrode ( 22  cm ) was fixed on them , and an inactive electrode of the same size was fixed 
 on the abdomen and connected to an hvpcs device ( endomed 482 , enraf- nonius , rotterdam , 
 netherlands ) . the same pulse frequency and pulse duration were established for application 
 of hvpcs to each group . a non - visible contraction intensity of 25 to 65 v was applied to the 
 experimental group i , and a visible contraction intensity of 40 to 85 v was applied to the 
 experimental group ii thirty minutes per day , for six days . placebo stimulation was applied 
 to the control group under the same condition as the experimental groups . for the first three days , 
 the cathode was applied as the active electrode , and from the fourth day , the anode was 
 applied as the active electrode20 . fibroblasts move to the cathode due to their electrotropism24 , and when the active electrode for hvpcs is the cathode , cellular 
 responses of fibroblasts are activated25 . the diameters of wounds were measured right after their induction , on the third day , on the 
 fifth day , and on the seventh day with the naked eyes using calipers ( mitutoyo corporation , 
 kawasaki , japan ) capable of measuring length in 0.05  mm increments . in order to compare the 
 sizes of wounds , the sizes of the left and right wounds were added and divided by two ( fig . 1fig . wound diameter measured with calipers western blotting was conducted in order to examine the expression of tgf-1 after 
 application of hvpcs . the wound tissues treated according to each manipulation condition 
 were washed three times with phosphate - buffered saline at 4  c cut finely lysis buffer 
 ( 300  mm nacl , 50  mm tris cl , ph 7.6 ) with a volume three times larger than that of the 
 tissues , 0.5% triton x100 , 2  mm phenylmethylsulfonyl fluoride , 2 l / ml aprotinin , and 2 
 l / ml leupeptin were added at 4  c to react for 40 minutes . thereafter , they were 
 centrifuged at 4  c and 14,000  rpm for 15 minutes to extract supernatant fluids . protein 
 quantification was performed with the supernatant fluids , and the extracted proteins were 
 distributed to 7.512% sodium dodecyl sulfate ( sds ) polyacrylamide gel for 
 electrophoresis26 . then blotting was 
 performed on a polyvinylidene difluoride membrane ( amersham , 0.2 m , pore size , usa)27 . the membrane was reacted for one hour at 
 4  c in 5% ( w / v ) nonfat dried milk containing triethanolamine - buffered saline ( tbs , 
 phosphate - buffered saline containing 0.01% ( v / v ) tween 20 ) and washed with tbs . tgf-1 
 ( sc-146 , 1:500 , santa cruz biotechnology , santa cruz , ca , usa ) was reacted for 24 hours at 
 4  c , and to verify whether there was a specific reaction and the degree of the reaction , a 
 goat anti - rabbit igg antibody ( ab6721 , abcam , cambridge , ma , usa ) was reacted as the 
 secondary antibody for one hour ; an enhanced chemical luminescence kit ( rpn 2106 , amersham 
 life science , inc . , usa ) was used to determine the results . to examine whether the same 
 amount of protein was loaded , monoclonal anti--actin ( a-5316 , 1:5,000 , sigma - aldrich , st , 
 louis , mo , usa ) and goat anti - mouse igg ( bd bioscience , franklin lakes , nj , usa ) antibodies 
 were used for comparison . after application of hvpcs , immunohistochemical assessment was conducted in order to look 
 at expression of type i collagen . after the paraffin removal and hydration processes for the 
 produced slide , 30% tris - ethylenediaminetetraacetic acid ( edta ) was reacted for 40 minutes 
 at 90  c and cooled for 20 minutes at a room temperature in order to remove nonspecific 
 reactions for immunohistochemistry . then as a preprocessing step , 3% hydrogen peroxidase was 
 used to block the activity of endogenous peroxidase . thereafter , a pbs solution to which 
 blocking serum had been added was used so that the primary antibody solution would be well 
 absorbed into the tissue inside . the tissue was then cleaned with 0.01  m pbs several times , 
 and the primary antibody for type 1 collagen ( sigma - aldrich , st . louis , mo , usa ) was diluted 
 1:100 , 1:200 , 1:500 , and 1:2000 with pbs . it was reacted over night at 4  c and then washed 
 with pbs . then , the universal antibody was diluted with blocking serum in pbs and reacted 
 for 60 minutes at a room temperature . again , the tissue was washed with 0.01  m pbs three 
 times , for five minutes each time and reacted with streptavidin for 30 minutes at room 
 temperature . after washing with pbs , color development was performed with dab 
 ( 3,3-diaminobenzidine , d3939 , sigma - aldrich , st . then , it 
 was washed with pbs once for three minutes , counterstained with hematoxylin , washed twice 
 for three minutes in flowing water , and dehydrated with 70% , 80% , 90% , and 100% ethanol for 
 two minutes for each . the tissue was treated with 100% xylene twice for two minutes to 
 render it transparent and then sealed with canada balsam ( sigma - aldrich , st . louis , mo , usa ) 
 to produce permanent samples . for statistical analysis of this study , pasw statistics , ver 18.0 , was used . in order to 
 verify the significance of each group s wound size and expression of tgf-1 according to 
 intensity of hvpcs , one - way analysis of variance ( anova ) was conducted to examine 
 differences among the groups at each measured time point , and duncan s test was performed as 
 a post hoc test . a paired t - test was carried out in order to look at each group s 
 differences in measured variables at each measured time point . immunochemical assessment of type i collagen was performed in a 
 semiquantitative manner and classified as follows : negative reaction , mild negative reaction 
 when stainability was weak , moderate positive reaction when stainability was moderate , and 
 strong positive reaction when stainability was strong28 . according to the results of comparing changes in wound size at each time point , the control 
 group s wound size was significantly decreased on the fifth and seventh days ( p<0.01 ) , 
 while the wound sizes of experimental groups i and ii were significantly decreased at all 
 time points ( p<0.05 ) . according to the results of one - way anova for comparison among the 
 groups , there was significant difference on the fifth and seventh days , and therefore , the wound size of experimental group ii was the most 
 significantly decreased on the fifth and seventh days ( p<0.01 ) ( table 1table 1.comparison of changes in wound size ( unit :  mm)day 0day 3day 5day 7control group ( n=12)10.5  0.39.9  1.08.6 1.03.74  0.3f2.14.019.422.0post hoca , b > ca > b > cvalues are presented as the meansd . tested by one - way anova ( p<0.05 ; 
 p<0.01 ) and duncan s multiple range test . tested by paired t - test 
 ( * p<0.05 ; * * p<0.01 ) . a. control group : sham group ( wound induced ) . b. 
 experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental 
 group ii : 4085v , hvpcs . ) . values are presented as the meansd . tested by one - way anova ( p<0.05 ; 
 p<0.01 ) and duncan s multiple range test . tested by paired t - test 
 ( * p<0.05 ; * * p<0.01 ) . b. 
 experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental 
 group ii : 4085v , hvpcs . according to the results of comparing the expressions of tgf-1 at each time point , the 
 expression of tgf-1 in the experimental groups was significantly increased on the fifth day 
 ( p<0.05 ) . according to the results of one - way anova to compare the expression of tgf-1 
 among the groups , there were significant differences on the fifth day , and therefore , 
 duncan s post hoc test was conducted . the expression of tgf-1 in experimental group ii was 
 highest the fifth day ( p<0.01 ) ( table 
 2table 2.comparison of changes in tgf -1 expression ( unit : % ) day 3 day 5day 7control group ( n=12)99.28.199.05.799.59.7experimental group i ( n=12)105.78.1130.212.1101.28.8experimental group ii ( n=12)105.77.9146.611.4 * 117.010.7f0.822.53.8post hocc > b > avalues are presented as the meansd . tested by one - way anova ( p<0.05 ; 
  p<0.01 ) and duncan 's multiple range test . tested by paired t - test 
 ( * p<0.05 ; * * p<0.01 ) . a. control group : sham group ( wound induced ) . b. 
 experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental 
 group ii : 4085v , hvpcs . ) . values are presented as the meansd . tested by one - way anova ( p<0.05 ; 
  p<0.01 ) and duncan 's multiple range test . tested by paired t - test 
 ( * p<0.05 ; * * p<0.01 ) . b. 
 experimental group i : 2565v , high voltage pulsed current ( hvpcs ) . c. experimental 
 group ii : 4085v , hvpcs . according to the results of observation of the immunoreactivity to type i collagen , which 
 is known to be a component making up most of the internal structure of a wound , the control 
 group and experimental group i showed a mild positive reaction ( + ) on the fifth day , and 
 experimental group ii showed a strong positive reaction ( + + + ) in the dermis on the fifth day 
 and a moderate positive reaction ( + + ) on the seventh day ( table 3table 3.immunohistochemical reaction of type i collagen of the skingroupperiod3 days5 days7 dayscontrol group -+-experimental group i-+-experimental group ii-+++++ and figs . 2fig . 2.immunohistochemical findings for the type i collagen reaction in skin from the 
 control group ( immunohistochemical stain , 100 ) , 3fig . 3.immunohistochemical findings for the type i collagen reaction in skin from 
 experimental group i ( immunohistochemical stain , 100 ) , 4fig . 4.immunohistochemical findings for the type i collagen reaction in skin from 
 experimental group ii ( immunohistochemical stain , 100 ) ) . immunohistochemical findings for the type i collagen reaction in skin from the 
 control group ( immunohistochemical stain , 100 ) immunohistochemical findings for the type i collagen reaction in skin from 
 experimental group i ( immunohistochemical stain , 100 ) immunohistochemical findings for the type i collagen reaction in skin from 
 experimental group ii ( immunohistochemical stain , 100 ) hvpcs has a monophasic twin peak pulse wave form , low voltage of 150 v or higher , low total 
 current ( 1.5 ma ) , and very short pulse duration29 . the effects of hvpcs include a decrease in chronic decubitus 
 ulcer , inhibition of staphylococcus aureus growth , an increase in perfusion around ischemic 
 wounds , and improved microcirculation15,16,17,18 . this study aimed to examine expression of type i collagen and tgf-1 by applying hvpcs with 
 a visible contraction intensity to wound - induced white rats . the same pulse frequency and 
 duration were set for application of hvpcs to each group . a non - visual contraction intensity 
 of 25 to 65 v was applied to experimental group i , and a visual contraction intensity of 40 
 to 85 v was applied to experimental group ii ; both groups received hvpcs for thirty minutes 
 per day for six days . according to the results of comparing changes in wound size at each 
 time point , the control group saw a significant decrease in its wound size on the fifth and 
 seventh days ( p<0.01 ) , while experimental groups i and ii saw a significant decrease in 
 their wound sizes at all time points ( p<0.05 ) . wound size was compared among the groups , 
 and that of the experimental group ii was more significantly decreased than the other groups 
 on the fifth and seventh days ( p<0.01 ) jeong and song applied hvpcs 
 with a non - visible contraction intensity ( 140  s , 120 pps , and 3050 v ) to normal white rats 
 30 minutes per day for seven days and reported that expression of tgf-1 in the dermis 
 significantly increased relative to the control group20 . however , western blotting was conducted in the present study to 
 compare the expression of tgf-1 in each group at each time point . the expression of tgf- 1 
 in experimental group ii was significantly increased on the fifth day ( p<0.05 ) , which was 
 more or less different from previous study results . according to the results of comparing 
 the expression of tgf-1 among the groups at each time point , the expression of tgf-1 in 
 experimental group ii this result 
 is considered to have been caused by the relatively high stimulation intensity , which 
 triggers contraction of nerve roots as a result of the pumping action of the muscles and 
 increased local vasodilatation and oxygen supply to tissues32 , 33 , thereby stimulating 
 endothelial blood vessel cells and promoting formation of blood vessels , which in turns 
 induces stimulation of macrophages and platelets , resulting in increased expression of 
 tgf-17 . according to the results of the immunohistochemical reaction performed to examine the 
 expression of type i collagen , the control group and experimental group i showed a mild 
 positive reaction ( + ) on the fifth day , and experimental group ii showed a strong positive 
 reaction ( + + + ) in the dermis on the fifth day and a moderate positive reaction ( + + ) on the 
 seventh day . richard et al . observed that wounds were in the inflammatory phase on the third 
 day after injury and that they were in the remodeling phase on the sixth day , with the 
 inflammatory stage almost over and granulation tissue starting to form34 . this result shows that synthesis of type i collagen is 
 most active on the fifth day and that synthesis of type i collagen is promoted by hvpcs with 
 a visible contraction intensity . lee et al . also observed 
 that fibroblast expression induced collagen synthesis10 . in the present study as well , it is judged that tgf-1 increased 
 by hvpcs with a visible contraction intensity promoted type i collagen synthesis . comparison of the sizes of the wounds among the groups showed that the most significant 
 decrease in wound size was found in experimental group ii on the fifth and seventh days . 
 according to the result of comparison of the expression of tgf-1 in each group at each time 
 point , the expression of tgf-1 in experimental group ii was significantly increased on the 
 fifth day . according to the results of the immunohistochemical reaction performed to examine 
 the expression of type i collagen , the control group and experimental group i showed a mild 
 positive reaction ( + ) on the fifth day , and experimental group ii showed a strong positive 
 reaction ( + + + ) in the dermis on the fifth day and a moderate positive reaction ( + + ) on the 
 seventh day . the above results suggest that hvpcs with a visible contraction intensity 
 increased expression of tgf-1 and synthesis of type i collagen , promoting wound 
 healing .