regeneration of injured tibialis anterior muscle in mice

Original Article J. St. Marianna Univ.Vol. 6, pp. 159–169, 2015

1 Department of Sports Medicine, St. Marianna University Graduate School of Medicine2 Department of Sports Medicine, St. Marianna University School of Medicine3 Department of Physiology, Graduate School of Health Sciences, Toyohashi SOZO University4 Department of Physiology, St. Marianna University School of Medicine5 Department of Orthopaedic Surgery, St. Marianna University School of Medicine

Regeneration of Injured Tibialis Anterior Muscle in Mice in Response to

Microcurrent Electrical Neuromuscular Stimulation with or without Icing

Atsuhiro Yoshida1, Hiroto Fujiya1, 2, Katsumasa Goto3, Mitsutoshi Kurosaka4, Yuji Ogura4, Kanaka Yatabe2, Hirotaka Yoshioka1, Koh Terauchi1, Toshiya Funabashi4,

Tatsuo Akema4, Hisateru Niki5, and Haruki Musha1, 2

(Received for Publication: August 7, 2015)

AbstractObjective: Cold therapy, so-called icing, is often used immediately after muscle injuries as first aid for the sup‐pression of inflammation and pain relief. Recent evidences show icing retards the regeneration of injured skele‐tal muscle. On the other hand, microcurrent electrical neuromuscular stimulation (MENS) promotes the regen‐eration of injured skeletal muscle. In this study, we investigated the effects of the MENS with or without icingon the regeneration of injured skeletal muscle.Methods: Eight-week-old male mice (C57BL/6J) were divided into 4 groups: control (C), cardiotoxin (CTX)injected (X), CTX injected with MENS (XM), and CTX injected with combined treatments with icing andMENS (XIM) groups. Necrosis-regeneration cycle was induced by an intramuscular injection of CTX into tibia‐lis anterior (TA) muscles except for C group. After CTX–injection, the hindlimbs of the mice were soaked inice-cold water (4ºC) for 20 minutes under anesthesia (XIM). After the treatments, both right and left hindlimbsof the mice in XIM and XM groups were treated with MENS (10 µA, 0.3 Hz, 250 msec) for 60 min a day and 3days per week for 1 or 3 weeks. One and three weeks after CTX injection, TA muscles were dissected.Results: MENS with or without icing facilitated the recovery of muscle protein content and muscle fiber mor‐phology including mean fiber cross-sectional areas of injured TA muscle, compared with non-treated condition.These facilitating effects of MENS with or without icing were accompanied with the increase in the relativenumber of Pax7-positive nuclei, namely satellite cells. Judging from fiber morphology, MENS with icing hadenhanced stimulating effects on the regeneration of injured skeletal muscle, compared with MENS-treated con‐dition.Conclusion: Evidence suggested that MENS with or without icing facilitated the regeneration of injured TAmuscle. A combination treatment of MENS with icing might be a useful therapy for sports-related skeletal mus‐cle injuries.

Key wordsmuscle injury, microcurrent electrical neuromuscular stimulation, icing, satellite cell

Introduction

Skeletal muscle injuries are common sports-re‐lated injuries, and several months are required for re‐

turn to normal function and activity1)2), causing con‐cern for athletes. A great deal of clinicians andresearchers have focused on the promotion of repairand regeneration in injured skeletal muscle3–8). Non-

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pharmacological treatments including heat and coldtherapies have been applied to facilitate the regenera‐tion of injured skeletal muscle. Cold therapy, so-called icing, is often used immediately after the in‐jury as first aid9)10). Icing is generally considered to beuseful for pain relief and anti–inflammation (swellingand bleeding)11–14). On the other hand, both positiveand negative effects of icing on skeletal muscle injuryhave been reported14)15). Although icing suppressesinflammation12)16), it delays muscle regeneration17)18).Therefore, even though icing is considered essentialfor first aid to reduce swelling and pain immediatelyafter muscle injury, the physiological effects of icingon injured skeletal muscle are controversial19).

Microcurrent electrical neuromuscular stimula‐tion (MENS) has been used as one of the treatmentmethods for sports-related skeletal muscle injury20).Results suggest that MENS offers pain relief21) andpromotes soft tissue repair. Furthermore, it has beenalso reported that MENS facilitates the regenerationof tendon injuries22)23), skin ulcers24)25), wounds26)27),bedsores28), ligament injuries29), and skeletal muscleinjuries20).

Considering the effects of both therapies, a com‐bined therapy of MENS with icing may be useful forthe regeneration of injured skeletal muscle. However,there is no evidence for the effects of the combinedtherapy. In the present study, we investigated the ef‐fects of MENS with or without icing on the regenera‐tion of injured skeletal muscle.

Materials and Methods

AnimalsAll experimental procedures were carried out in

accordance with the Guide for the Care and Use ofLaboratory Animals as adopted and promulgated bythe National Institutes of Health (Bethesda, MD) andwere approved by the Animal Experiment Committeeof the Experimental Animal Research Facilities, St.Marianna University School of Medicine.

Forty-eight 7-week-old male C57BL/6J mice(Japan SLC Inc., Hamamatsu, Japan) were used.Mice were housed in an environmentally controlledroom (24 ± 1 oC and 55 ± 10% relative humidity)with a 12/12 hour light-dark cycle. Animals were fedstandard rat chow and water ad libitum. After a weekof acclimation to the animal facility, mice were ran‐domly divided into 4 groups: control (C), cardiotoxin(CTX) injected (X), CTX injected with MENS (XM),and CTX injected with a combined treatment of icingand MENS (XIM) groups (n=12 in each group).

Skeletal muscle injury modelThe necrosis-regeneration cycle was induced by

an intramuscular injection of CTX (100 µl, 10 µM insaline, Latoxan, France) into the proximal, middle,and distal regions of both the right and left tibialis an‐terior (TA) muscles except for C group, using a 27-gauge needle under anesthesia with the mice breath‐ing isoflurane as described earlier30–32). The samevolume of physiological saline was injected into bothright and left TA muscles of mice in C group. Thisprocedure for the initiation of necrosis-regenerationwas performed carefully to avoid the damage in thenerves and blood vessels, as was suggested else‐where33)34).

Icing treatmentAfter CTX–injection, both right and left hin‐

dlimbs of mice were completely soaked in ice-coldwater (4ºC) for 20 minutes under anesthesia with iso‐flurane in the XIM groups as described previously18).During icing, the abdomen of each mouse was wrap‐ped with an expanded polystyrene board and the tailwas clipped to the back to avoid direct contact withcold water. In a pilot study, time course of the tem‐perature of TA muscle and the esophagus in micewere monitored with a thermocouple thermometerPTW-301 (Unique Medical Co., Ltd., Tokyo, Japan).The muscle temperature fell to ~4ºC immediately af‐ter icing, and was maintained for 20 min. On theother hand, the esophageal temperature became~30ºC approximately 15 minutes after the initiationof icing.

Microcurrent electrical neuromuscular stimulation(MENS)

After icing treatment, both right and left hin‐dlimbs of mice in XIM and XM groups were treatedwith MENS (10 µA, 0.3 Hz, 250 msec) using an elec‐trical stimulator (Trio300, Ito Co., Ltd., Tokyo, Ja‐pan) for 60 min a day and 3 days per week for 1 or 3weeks under an anesthesia with isoflurane as descri‐bed in a previous study20) (Figure 1). Mice of Xgroup also received the anesthesia without MENS.Before MENS treatment, the epilation of both rightand left hindlimbs of mice in all groups were per‐formed using a commercial hair remover. Then, twoelectrodes were placed on the distal anterior side ofthe knee joint and the proximal anterior side of theankle joint, respectively.

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Yoshida A Fujiya H et al160

Figure 1. Experimental protocol.

C: control group, X: cardiotoxin (CTX) -injec‐

ted group, XM: CTX–injected with MENS treat‐

ment group, XIM: CTX–injected with combined

treatments with icing and MENS group. 1 week:

one week after CTX injection, 3 weeks: three

weeks after CTX injection.

▽: CTX injection, ▲: icing, ●: sampling, ↑:

MENS (dotted line).

SamplingOne and three weeks after CTX injection, mice

in all groups were sacrificed under anesthesia withisoflurane. TA muscles were dissected from both hin‐dlimbs and rapidly weighed (wet weight) and dividedinto three portions cross-sectionally. Then, musclesections were frozen in isopentane cooled by liquidnitrogen and stored at -80ºC until analysis (Figure 1).

Histological and immunohistochemical analysesSerial transverse cryosections (10-µm thick) of

the frozen central portion of TA muscles were cut at-20oC using a cryostat (CM1900, Leica, Wetzlar, Ger‐many) and mounted on the slide glasses. The sectionswere air-dried and stained to evaluate the histologicalstage using hematoxylin and eosin (HE), and the pro‐files of Pax7-positive nuclei by the standard immuno‐histochemical technique35).

Since Pax7 is a specific marker for muscle satel‐lite cells36–39), cryosections were stained with mousemonoclonal anti-chicken Pax7 (1:5, Developmentalstudies Hybridoma Bank, Iowa, IA, USA). Cryosec‐tions were fixed with paraformaldehyde (4%) at 4ºCfor 15 minutes. After blocking for 7 minutes using areagent (Large Volume Ultra V Block, Thermo Sci‐entific, Cheshire, UK), samples were incubated withthe primary antibodies for Pax7 and rabbit polyclonalanti-human laminin (1:1000, Z0097, Dako Cytoma‐tion, Glostrup, Denmark) at room temperature for 3hours. Laminin immunohistochemical staining was

performed to detect basement membrane30)31)35)40)41).Sections were incubated with secondary antibodies,Alexa Fluor 568-labeled goat anti-mouse IgG1(1:500; Invitrogen, Molecular Probes, Eugene, OR,USA) and Alexa Flour 488-labeled goat anti-rabbitIgG (1:1000; Invitrogen, Molecular Probes, Eugene,OR, USA). Then, nuclei were stained by a 5 -minuteincubation in a solution of 4',6-diamidino-2-phenylin‐dole (DAPI, 1:10000; Sigma-Aldrich, St Louis, MO,USA) (Figure 2).

Stained slides were viewed using a KEYENCEBZ-9000 microscope (KEYENCE, Osaka, Japan) andimages were captured using image software (KEY‐ENCE). The cross-sectional areas (CSAs) of fibershaving central nuclei of 250–500 muscle fibers ineach muscle were calculated using Image J (Ver.1.45i, Wayne Rasband, National Institutes of Health,USA). The percentage of fibers having central nucleirelative to total muscle fibers in the whole transversesection of 250–500 muscle fibers was also calculated.Pax7-positive nuclei located within laminin-positivebasal membrane were counted in the whole trans‐verse section of 250–500 fibers. The number of myo‐nuclei and Pax7-positive nuclei per muscle fiber werecalculated as described previously42).

Muscle protein contentA part of the distal portion of TA muscles were

re-weighted and homogenized in ice-cold RIPA (Ra‐dio-Immunoprecipitation Assay) buffer (ThermoFisher Scientific, Waltham, MA, USA), and thencompletely solubilized by alkaline treatment with 2 NNaOH at 37°C for 1 hour. Protein concentrations ofhomogenate samples were measured using a BCAprotein assay kit (PIERCE, Pittsburgh, PA, USA) andbovine serum albumin as the standard. Protein con‐tent per milligram of TA muscle wet weight was cal‐culated, and then absolute protein content in thewhole muscle was calculated using the whole TA wetweight, according to a previous study35).

Western blottingThe remaining portion of TA muscle was homo‐

genized on ice with RIPA buffer (Thermo Fisher Sci‐entific, MA, USA) supplemented with protease andphosphatase inhibitors (Roche Diagnostics, Man‐heim, Germany). Proteins were loaded onto 10%SDS-PAGE gels and run at 200 V for 30 minutes.Following SDS-PAGE, proteins were transferred ontonitrocellulose membranes using a Bio-Rad MiniTrans-Blot cell at 100 V for 60 min at 4°C. Then, the

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MENS with icing for muscle injury 161

Figure 2. Identification of satellite cell and myonuclei.

Arrows indicate Pax7-positive nuclei outside the laminin.

a: Pax7, b: laminin, c: DAPI, d: merged.

membranes were blocked with 5% nonfat dry milk inTris-buffered saline with 0.1% Tween (TBST). Afterblocking, membranes were incubated with the pri‐mary antibodies: rabbit polyclonal anti-mouse phos‐phorylated Ser473- Akt (1:2000; Cell Signaling,#9271, Beverly, MA, USA) and rabbit polyclonalanti-mouse Akt (1:2000; Cell Signaling, #9272), andthen reacted with rabbit horseradish peroxidase-con‐jugated secondary antibodies (1:5000; Cell Signaling,#7074) for 1 hour at room temperature. After severalwashes, the protein bands were visualized usingchemiluminescence regent (ECL Plus Western blot‐ting detection reagents: GE Healthcare, Tokyo, Ja‐pan) and captured with a LAS-3000 imaging system(Fuji film, Tokyo, Japan). The signal density of theprotein bands was evaluated using Image J (Ver.1.45i).

Statistical analysesAll values were expressed as means ± SEMs.

Statistical significance for each measurement was an‐alyzed by using two-way (treatment x time) ANOVA.When a significant interaction between two main fac‐tors was observed, Tukey post hoc test was carriedout. The significant level of Akt expression was tes‐ted by Student t-test. All of the statistical analyses

were performed using SPSS Statistics 20.0J (IBM Ja‐pan, Tokyo, Japan). The significance level was accep‐ted at p<0.05.

Results

Muscle wet weight and protein contentMuscle wet weight relative to body weights one

and three weeks after CTX injection are shown inFigure 3. At both one and three weeks, significantmain effects (treatment and time) and interactions(treatment × time) were observed (p<0.05). Relativemuscle wet weight in X and XIM groups were signif‐icantly lower than that in C group one week after theinjection. Three weeks after CTX injection, musclewet weight in X, XM, and XIM groups were signifi‐cantly higher than that in C group (p<0.05). However,there was no significant difference in muscle wetweight among CTX–injected groups.

There were significant main effects (treatment)and interactions (treatment × time) in muscle proteincontent one and three weeks after CTX injection(Figure 4, p<0.05). Muscle protein content in Xgroup significantly decreased, compared with groupC (p<0.05). Three weeks after the injection, therewas no significant difference in muscle protein con‐tent among the 4 groups.

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Figure 3. Muscle wet weight relative to body weight.

Values are means ± SEMs. n=6 in each group.

a and b: significant main effects of treatment (a) and time (b) (p<0.05, analyzed by two-

way ANOVA (treatment x time), c: significant interaction between two main effects

(p<0.05) analyzed by two-way ANOVA (treatment x time), *: p<0.05

Abbreviations are the same as in Figure 1.

Figure 4. Muscle protein content relative to body weight.


Abbreviations are the same as in Figures 1 and 3.

Morphology of muscle fibersFigure 5 shows the morphological responses of

CTX–injected muscles. One week after the injection,infiltrating cells were noted in X and XM groups(Figure 5A). In XIM group, however, many regener‐ating fibers with central nuclei were observed. In Xand XM groups, regenerating fibers with a small di‐ameter and central nucleus were seen even after 3weeks, compared with XIM group. Three weeks afterCTX injection, the number of fibers with a centralnucleus decreased and fiber diameter increased, com‐

pared with X and XM groups (Figure 5B).Mean CSAs of fibers having a central nucleus

are shown in Figure 6. There were significant maineffects (treatment and time) and interactions (treat‐ment × time) in the mean fiber CSAs one and threeweeks after the injection (p<0.05). One week afterthe injection, the number of fiber CSAs in X, XM,and XIM groups was significantly lower than that ingroup C (p<0.05). Furthermore, there were signifi‐cant differences in mean fiber CSAs between X andXIM, and XM and XIM, respectively (p<0.05). Three

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Figure 5. Transverse cryosections of midbelly region of mouse tibialis anterior muscle stained with

hematoxylin and eosin at 1W (A) and 3W (B).


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Figure 6. Fiber cross-sectional areas.



Figure 7. Population of Pax7-positive nuclei (satellite cell) relative to total myonuclei.



weeks after CTX injection, the number of mean fiberCSAs in X and XM groups was also significantlylower than that in C group (p<0.05). However, therewere no significant differences in mean fiber CSAsbetween C and XIM, and between XM and XIM.

Number of Pax7-positive nucleiFigure 7 shows the changes in the number of

pax7-postive nuclei relative to total myonuclei in re‐sponse to CTX injection. There were significant maineffects (treatment and time) and interactions (treat‐

ment × time) in the relative population of Pax7-posi‐tive nuclei (p<0.05). The relative number of Pax7-positive nuclei in X, XM, and XIM groups wassignificantly higher than that in C group one andthree weeks after CTX injection (p<0.05). One weekafter the injection, the relative number of Pax7-posi‐tive nuclei in XM and XIM groups was also signifi‐cantly higher than that in X group. There were signif‐icant differences in the relative number of Pax7-positive nuclei between X and XIM, and betweenXM and XIM groups at three weeks (p<0.05).

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Figure 8. Expressions of phosphorylated and total Akt.

p-Akt: phosphorylated Akt, t-Akt: total Akt.

The expression level of p-Akt and t-Akt was

evaluated one week after CTX injection.



†: p=0.059.

Akt expressionIn this study, the activation level of Akt was

evaluated using the phosphorylated level of Akt rela‐tive to the total expression form (p-Akt/t-Akt), sincephosphorylated form of Akt is enzymatically ac‐tive43)44). Figure 8 shows the relative expression levelsof p-Akt to t-Akt one week after CTX injection inXM and XIM groups. The relative expression level ofp-Akt in XIM was higher than that in XM group(p=0.059).

Discussion

In the present study, we investigated the effectsof MENS treatments with or without cold therapy onthe regeneration of injured TA muscle in mice. Re‐sults showed that MENS with or without icing facili‐tated the recovery of muscle protein content and mus‐cle fiber morphology including mean fiber CSAs ofinjured TA muscle, compared with non-MENS-treat‐ment. The population of Pax7-positive nuclei, namelysatellite cells, was increased by MENS with or with‐out icing. Judging from fiber morphology, MENSwith icing had enhanced stimulating effects on the re‐generation of injured skeletal muscle, compared withMENS without icing. The expression level of p-Aktin MENS with icing was also higher than that inMENS treatment without icing.

Effects of MENSMENS without icing facilitated the recovery of

muscle wet weight, muscle protein content, and meanfiber CSAs in injured TA muscle, compared withnon-treated X group. These results are consistentwith the previous study20). Although the molecularmechanism(s) of MENS-associated facilitation of in‐jured skeletal muscle remain unclear, the involvementof the active transport of amino acids through in‐creased generation of adenosine triphosphate (ATP)or elevated protein synthesis have been sugges‐ted45)46). It has been reported that electric current in‐creases ATP synthesis up to 500% at 500 µA in skintissue, whereas ATP synthesis gradually decreasedbeyond 1,000 µA47). Synthesis of ATP might be acause of the facilitations of injured skeletal muscle.

MENS also increased the relative number ofPax7-positive nuclei one week after CTX injection,compared with C group. A MENS-associated in‐crease in Pax7-positive nuclei was also observed inthe previous study20). The population of Pax7-positivenuclei increased in muscle following CTX–injection-associated injury31). In the present study, a CTX injec‐

tion-associated increase in the number of Pax7-posi‐tive nuclei was also observed. One week after theinjection, MENS increased the number of Pax7-posi‐tive nuclei, compared with non-treated group.MENS-associated facilitation of the regeneration ofinjured skeletal muscle might be attributed to thehigher level of Pax7-positive nuclei. However, themolecular mechanism(s) of MENS-associated in‐crease in Pax7-positive nuclei are unknown. Addi‐tional investigations should be needed to elucidatethis point.

Combined effects of MENS with icingIn this study, the combined effects of MENS

with icing were investigated. MENS with icing facili‐tated the regeneration of injured TA muscle, com‐pared with non-treated X group. These facilitating ef‐fects of MENS with icing were almost identical tothose of MENS alone. This is the first study to inves‐tigate the combined effects of MENS and icing on theregeneration of injured skeletal muscle. Recent evi‐dences show that icing suppressed the regeneration ofinjured skeletal muscle in rats17) and mice18). Al‐though the effects of icing alone on the regeneration

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of injured TA muscle was not investigated in thepresent study, icing may depress the regeneration ofinjured TA muscle. If so, MENS following icingmight diminish such negative effects on the skeletalmuscle regeneration.

Histological analyses show MENS with icingstimulated the regeneration of injured TA muscle,compared with MENS alone (Figure 5). One weekafter CTX injection, the activation level of Akt inMENS with icing was higher than that in MENSalone. Overexpression of activated Akt promotes theregeneration of CTX injection-associated injuredmuscle fibers48). Furthermore, three weeks after theinjection, a higher level of the population of Pax7-positive nuclei in MENS with icing was observed.Although these factors may be a part of the facilita‐tion of injured TA muscle, we have no clear explana‐tion for the reason why the combined treatment ofMENS with icing has such effects.

PerspectiveIcing on sports-related injuries is important for

both the suppression of inflammation and pain relief.Furthermore, icing-associated vasoconstriction de‐creases edema formation and cellular damage withhypoxia of injured tissue9)10). On the other hand, re‐cent evidence shows icing retards the regeneration ofinjured skeletal muscles17)18). Therefore, the combina‐tion treatment of MENS with icing might be a usefultherapy for sports-related skeletal muscle injurieswith both the suppression of inflammation and painrelief.

Conclusion

The present study investigated the effects ofMENS with or without icing on the regeneration ofinjured TA muscle in mice. Evidence suggested thatMENS with or without icing facilitated the regenera‐tion of injured TA muscle. A combination treatmentof MENS with icing might be a useful therapy forsports-related skeletal muscle injuries with both thesuppression of inflammation and pain relief.

Acknowledgments

This study was supported, in part, by the JapanSociety for the Promotion of Science (21500639, HF;24500802, HF; 15K01632, HF; 26350818, KG;26560372, KG), the Uehara Memorial Foundation(KG), the Naito Foundation (KG), and the GraduateSchool of Health Sciences, Toyohashi SOZO Univer‐sity (KG). KG received research funding from Ito

Co., Ltd., but the sponsor had no role in study design,data collection and analysis, decision to publish, orpreparation of the manuscript. There are no patents,products in development or marketed products to de‐clare. The authors have no conflict of interest.

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regeneration of injured tibialis anterior muscle in mice

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