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Effects of low-level laser therapy (LLLT) on the nuclear factor (NF)-κB signaling pathway in traumatized muscle

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Effects of low-level laser therapy (LLLT) on the nuclear factor (NF)-κB signaling pathway in traumatized muscle
  Lasers in Surgery and Medicine 38:704–713 (2006) Effects of Low-Level Laser Therapy (LLLT) on the NuclearFactor (NF)- k B Signaling Pathway in Traumatized Muscle Carem Fetter Rizzi,  MSc , 1 Jose´ Luis Mauriz,  PhD , 2 Daniela Sousa Freitas Correˆa,  MSc , 1  Andre´a Janz Moreira,  MSc , 1 Claudio Galeano Zettler,  MD ,  PhD , 1,3 Lidiane Isabel Filippin,  MSc , 4 Norma Possa Marroni,  PhD , 1,5 and Javier Gonza´lez-Gallego,  MD ,  PhD 2 * 1  Department of Physiotherapy and Physiology, Universidade Luterana do Brasil, Brazil  2  Department of Physiology, University of Leo´ n, Spain 3  Department of Pathology, Fundac¸a˜  o Faculdade Federal de Cieˆ ncias Me´ dicas, Brazil 4  Laboratory of Experimental Hepatology and Physiology, Hospital de Clı´ nicas de Porto Alegre, Brazil 5   Department of Physiology, Universidade Federal de Rio Grande do Sul, Brazil BackgroundandObjective: Toinvestigatetheeffectsof low-level laser therapy (LLLT) on nuclear factor kappa B(NF- k B) activation and inducible nitric oxide synthase(iNOS) expression in an experimental model of muscletrauma. Study Design/Materials and Methods:  Injury to thegastrocnemius muscle in the rat was produced by a singleimpactblunttrauma.Alow-levelgaliumarsenide(Ga–As)laser (904 nm, 45 mW, and 5 J/cm 2 ) was applied for35 seconds duration, continuously. Results:  Histological abnormalities with increase incollagenconcentration, and oxidative stress wereobservedaftertrauma.ThiswasaccompaniedbyactivationofNF- k Band upregulation of iNOS expression, whereas proteinconcentration of I k B a  decreased. These effects were block-ed by LLLT. Conclusion:  LLLT reduced the inflammatory responseinduced by trauma and was able to block the effects of reactiveoxygen species (ROS)release and the activationof NF- k B. The associated reduction of iNOS overexpressionand collagen production suggest that the NF- k B pathwaymay be a signaling route involved in the pathogenesis of muscle trauma. Lasers Surg. Med. 38:704–713, 2006.  2006 Wiley-Liss, Inc. Keywords:  oxidative stress; Ga–As laser; LLLT; muscletrauma; iNOS; NF- k B INTRODUCTION Muscle injuries are frequent both in sports and in theworkplace; more than 30% of the injuries seen in theclinician’s office are related to skeletal muscle [1]. Theseinjuries can occur via a variety of mechanisms, including those arising through direct trauma (e.g., laceration andcontusion) and those by indirect trauma (e.g., ischemia,denervation, and strain) [2], but the general process of muscle repair is similar in most cases [3,4]. The complex muscle healing process comprises severalphases, including degeneration, inflammation, regenera-tion, fibrosis/scar formation, and remodeling phase. For atherapeutic modality to be effective it should reduce theacute inflammatory response and accelerate the recoveryphase, so that regeneration rather than scar formation isenhanced[5].Duringtheinflammatoryphase,factorssuchas phagocytic stimuli increase the generation of reactiveoxygenspecies(ROS),leadingtooxidativestress[6,7].Over the last few years, a number of studies have providedevidence of an important role of ROS in mediating themicrovascular disturbance that follows soft tissue injury[8].ROSmayinflictdirectdamagetovitalcellconstituents,such as lipids, proteins, and DNA  [9], and also activate thetranscriptionfactornuclearfactorkappaB(NF- k B)which,inturn,increasestheexpressionoftheinducibleisoformof nitric oxide synthase (iNOS) and the synthesis of nitricoxide [10,11]. Indiscriminated destruction of cells andtissues by nitric oxide and its reactive nitrogen intermedi-ates may play a significant role in the pathology of manyinflammatory conditions [12].The regenerative phase begins approximately 1 weekpost-injury, peaks over the subsequent week, and thensteadilydeclines[2].Iftheregenerativephasewereallowedto proceed uninterrupted, the muscle would most likelyheal without scarring. However, this phase ends prema-turelyduetothesimultaneousproductionoffibroustissue. Although there are reports showing that effects of laserphotostimulation on wound healing may involve thepromotionofextracellularmatrixproductionanddegrada-tion [13,14], replacement of normal tissue by connective tissue, mediated by an excessive fibrosis, which maycompromise the functional capacity of tissues and providequalified healing, excessive collagen/extracellular matrixdeposition, and scar formation must be limited [15]. A relationship between oxidative stress and fibrosis hasbeen reported in different pathologies [16 – 18], and lipo- peroxidation seems to be involved in the regulation of  *Correspondence to: Javier Gonza´lez-Gallego, MD, PhD,DepartmentofPhysiology,UniversityofLeo´n,24071-Leo´n,Spain.E-mail: dfijgg@unileon.es Accepted 27 April 2006Published online 23 June 2006 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/lsm.20371   2006 Wiley-Liss, Inc.  collagen  a 1 (I) gene expression [19,20]. In addition, NF- k Bhas binding sites for promoters or enhancers of manycollagen formation associated genes, such as IL-6, mono-cyte chemo-attractant protein-1, or iNOS [21 – 23], and a relationship ofiNOS to collagenformation has beenshownin various wound models [24]. Although several authorshave demonstrated the potential of low-level laser therapy(LLLT) in the facilitation of the muscle healing process[25 – 27], LLLT effects on ROS production are still con- troversial. It has been suggested that LLLT could enhanceROS production activity of human neutrophils by theactivation of the superoxide converting system [28], andthereisareportthatLLLTirradiationenablesamorerapidactivation of the superoxide production system [29].However, laser irradiation has been demonstrated toreduce oxidative stress in different situations such asisolated human neutrophils, during abdominal surgery,in vitro in liposome membranes, and in an animal model of tendon healing  [15,30 – 33]. It is unknown, however, if  during wound healing effects on fibrosis and oxidativestress are accompanied by changes in the NF- k B pathway.Ratsprovideanexcellentmodelforevaluatingtheeffectsof laser on muscle healing because it is relatively simple toinduce injury and to apply the treatment under controlledconditions and environments. The present study wasdesigned to investigate whether collagen formation isassociated to oxidative stress, NF- k B activation, and iNOSoverexpression in an experimental model of rat trauma-tized gastrocnemius muscle and whether these effects areprevented by galium arsenide (Ga–As) LLLT. MATERIALS AND METHODS Animals Male Wistar rats weighing 250–300 g were used. Theanimals werecagedat22 8 C,with 12hourslight-dark cycleand free access to food and water until the time of experiments. All experiments were performed in accor-dance with the  Guiding Principles for Research Involving Animals  (NAS). Experimental and Irradiation Procedures  Animals were first randomly divided into three groups( n ¼ 30 per group) named as: control (C), trauma (T),trauma þ LLLT (TL).Gastrocnemius injury was induced by a single impactblunttraumainapressdevelopedbytheCentroIndustrialde Equipamentos de Ensino e Pesquisa Ltda (CIDEP/RS,Brazil) using the procedure of Lech et al. [34]. Briefly,injury was produced by a metal mass (0.459 kg) falling through a metal guide from a height of 18 cm. The impactkinetic energy delivered was 0.811 J (Fig. 1). During theprocedure rats were anesthetized with ketamine chlorhi-drate (Ketalar, Parke Davis, 100 mg/kg) and xilazine 2%(Rompun, Bayer, 50 mg/kg) cocktail i.p. Control rats werealso anesthetized to ensure standardization, but withoutmuscle trauma.The animals of the control (C) and trauma (T) groupswere not submitted to irradiation. A low-level Ga–As laserwas applied daily with a 904-nm wavelength, 45-mW average power, 5-J/cm 2 dosage, for 35 seconds duration,continuously, to animals of the trauma þ LLLT (TL) group.Lasertreatmentwasapplieddaily,underartificiallight,atthe same time by 7 days or 14 days. The laser was applieddirectly to the previously shaved skin by transcutaneousapplication, and the spot size was 0.7 mm. Beam laserincidence angle was kept perpendicular (90 8 ) to theirradiation surface.Rats were killed at 12 hours, 7 days, or 14 days forbiochemical evaluation and histological analysis of mus-cles. The animals were anesthetized with ketaminechlorhidrate and xilazine 2% cocktail i.p. The gastrocne-mius muscle was rapidly removed from both legs, snap-frozen in liquid nitrogen, and stored at   80 8 C until Fig.1. Imageofthedeviceusedtoinducemuscletrauma.Injurywasproducedbyametalmass(0.459 kg) falling through a metal guide from a height of 18 cm. The impact kinetic energydelivered was 0.811 J. LLLT AND NF- k B IN INJURED MUSCLE 705  analysis. The entire surgical procedure took less than10 minutes. Histology  For histological examination, a piece of the muscle of allanimals was trimmed and fixed by immersion in 10%bufferedformalinfor24hours.Theblocksweredehydratedinagradedseriesofethanolandembeddedinparaffinwax.Serial 3- m m sections were stained with hematoxylin andeosin.Fivesectionsfromeachsamplewereanalyzedbytwoindependent pathologists who had no prior knowledge of the animal groups. Collagen Quantification CollagenwasassayedaccordingtoRojkindandGonza´lez[35]. The concentration of collagen was determined bymeasuring the hydroxyproline content in muscles samplesafter digestion with acid. Muscles samples (100 mg) wereplaced in ampoules, 2 ml of 6 N HCl was added, and thenthey were sealed and hydrolyzed at 100 8 C for 24 hours.Next,thesampleswereevaporatedat50 8 Cfor24hoursandresuspendedin3mlofsodiumacetate/citricacidbuffer,pH6.0; 0.5 g of activated charcoal was added, the mixture wasstirred vigorously, and then centrifuged at 5,000  g  for10 minutes. Hydroxyproline oxidation was initiated byadding1mlofchloramineT(0.05M).Themixturewaskeptfor 20 minutes at room temperature, and 2 M sodiumthiosulfate and 1 N sodium hydroxide were added to thereaction. The pyrrole-containing samples were incubatedwith Ehrlich’s reagent for 30 minutes and the absorbancewas read at 560 nm. TBARS Analysis Oxidative stress was determined by measuring theconcentration of aldehydic products (MDA) by thiobarbi-turicacidreactivesubstances(TBARS)[36].Theamountof aldehydic products generated by lipid peroxidation wasquantifiedbythethiobarbituricacidreactionusing3mgof protein per sample. Results were referred as TBARS. Thesampleswereincubatedat90 8 Cfor30minutesafteradding 500  m l of 0.37% thiobarbituric acid in 15% trichloroaceticacid, and then centrifuged at 4 8 C at 2,000  g  for 15 minutes.Spectrophotometric absorbance was determined in thesupernatant at 535 nm.  Western Blot Analysis  Western Blot analysis was performed on cytosolicextracts. Briefly, 100 mg of muscle tissue from each ratwashomogenizedusingaglass-Teflonhomogenizerin1mlice-cold homogenization buffer (Tris 10 mM, Sucrose0.25 M, EDTA 5 mM, NaCl 50 mM, sodium phosphate30 mM, NaF 50 mM, sodium orthovanadate 100  m M, DTT1 mM, PMSF 1 mM, and protease inhibition cocktail,pH 7.4) and centrifuged at 14,000 rpm for 15 minutes. Theresultant supernatant (cytosolic fraction) was harvested,the protein content determined by Lowry method [37].Samples of cytosolic fraction containing 20–75  m g of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9–14% acrylamide)and transferred to nitrocellulose. Non-specific binding wasblocked by pre-incubation of the nitrocellulose in phos-phate-bufferedsalinecontaining5%bovineserumalbuminfor1hour.Thenitrocellulosewasthenincubatedovernightat 4 8 C with polyclonal-specific antibodies (from goat)against rat iNOS (130 kDa) or I k B a  (36 kDa) (sc-847 andsc-8310, Santa Cruz Biotechnology, Santa Cruz, CA).Bound primary antibody was detected with HRP-conju-gated anti-goat antibody (DAKO, Glostrup, Denmark) andblots were developed using an enhanced chemilumines-cence detection system (ECL kit, Amersham Pharmacia,Little Chalfont, UK). Densitometry analysis of specificbands was performed by Scion Image software (ScionCorporation, Frederick, MD). Electrophoretic Mobility Shift Assay (EMSA) Nuclear extracts were prepared from gastrocnemiusmuscle as described previously [38] in order to study theactivation of the transcription factor NF- k B. A commercialdouble-stranded oligonucleotide (5 0 -AGTTGAGGGGAC-TTTCCCAGGC-3 0 ) purchased from Promega (Madison, WI) was briefly end-labeled by T4 polynucleotide kinase.The DNA-binding reaction was performed by mixing 10  m g of nuclear extract in incubation buffer (10 mM Tris-HClpH7.5,40mMNaCl,1mMEDTA,and4%glycerol)and1 m g poly (dI-dC). After 15 minutes on ice, the labeled oligonu-cleotide(30,000cpm)wasaddedandthemixtureincubatedfor 20 minutes at room temperature. For the competitionassay the nuclear extract (10  m g) was pre-incubated withhomologousunlabeledNF- k Boligonucleotidefor5minuteson ice, followed by the addition of   g - 32 P end-labeled NF- k Bprobe.Forcompetitionstudies,3.5pmolofunlabeled(cold)NF- k Boligonucleotide(ascompetitor)or3.5pmoloflabeledNF- k B oligonucleotide mutate (as non-competitor) weremixed 15 minutes before the incubation with the labeledoligonucleotide. All samples were electrophoresed througha 6% polyacrimide gel for 90 minutes at 150 V. The gel wasthen dried and autoradiographed at  80 8 C with intensify-ingscreensonBiomaxfilm(EastmanKodakCo,Rochester,NY) for a whole week. Densitometry analysis of specificbands was performed by Scion Image software (ScionCorporation, Frederick, MD). Statistical Analysis Results are expressed as mean values  SEM. The datawerecomparedbyanalysisofvariance(ANOVA);whentheanalysis indicated the presence of a significant difference,the means were compared with the Newmann Keuls test.Significancewasacceptedat  P < 0.05.Valueswereanalyzedusing the statistical package SPSS 13.0 (SPSS, Inc.,Chicago, IL). RESULTS Histological analysis of muscle sections of the traumagroup showed modifications of normal architecture, withangiogenesis, vasodilatation, inflammatory reaction, andextracellular matrix formation 12 hours after trauma, and 706 RIZZI ET AL.  increase of fibrosis at 7 days and 14 days after trauma. Administration of Ga–As laser markedly alleviated histo-logical abnormalities both at 7 days and 14 days post-trauma. No histological alterations were detected inmuscles of the control group (Fig. 2).Collagen concentration was increased by muscle traumaat 7 days and remained increased 14 days post-trauma( þ 117%and þ 108%,respectively).AdministrationofLLLTwas able to reduce the presence of collagen in traumatizedrats both at 7 days and 14 days (Fig. 3).Oxidative stress, measured by the cytosolic concentra-tionofTBARS,wasincreasedbytrauma( þ 122%and þ 97%at 7 days and 14 days, respectively). LLLT reduced theseincreases in traumatized rats treated both for 7 days and14 days post-trauma (Fig. 4).Figure 5 illustrates the effects of trauma and LLLTon iNOS expression measured by Western blot. Traumacaused a significant induction of iNOS protein levels at7 days and 14 days ( þ 75% and  þ 65%, respectively). Thisincrease was abrogated by LLLT.To study the effects on NF- k B-binding activity, musclenuclear extracts were analyzed by electrophoretic mobilityshift assay (EMSA). As shownin Figure6, traumainduceda marked activation of NF- k B 7 days and 14 days post-trauma ( þ 82% and  þ 80%, respectively). This activationwassignificantlypreventedinratswithLLLT.Sinceithasbeen well documented that activation of NF- k B correlateswithrapidproteolyticdegradationofI k B a [39],weassessedprotein levels of this protein. Trauma resulted in a signi-ficant decrease of I k B a  (  48% and   43% at 7 days and14 days, respectively) which was blocked by LLLT admin-istration (Fig. 7). DISCUSSION Present treatment modalities of muscle injuries includesurgical repair, rest, cryotherapy, termotherapy, electro-therapy, compression, elevation, immobilization, andNSAIDs. Optimal treatment has not yet been determinedbecausethesetraditionalmethodsusuallydonotpromotearapid enough recovery to satisfy patients and the recur-rence of injury is common, indicatingthat suchtreatmentsfail to provide full functional recovery and are likelyineffective to prevent the formation of permanent scar Fig. 2. Photomicrographies of muscle sections in the different groups. No alterations weredetected in muscles of control group (C). Muscle sections of animals of the trauma group (T)showed modifications of the normal architecture, with, angiogenesis (A), vasodilatation (V),extracellular matrix formation (E), and inflammatory reaction (I) 12 hours after trauma, andincreaseonfibrosis(F)at7daysand14daysaftertrauma.AdministrationofGa–Aslaser(TL)markedly alleviated histological abnormalities both at 7 days and 14 days post-trauma.[Figure can be viewed in color online via www.interscience.wiley.com.] LLLT AND NF- k B IN INJURED MUSCLE 707  tissue [2–4]. The use of LLLT on wound healing has beeninvestigated by different researchers [25–27,40], butchanges induced in potential mediators of inflammationand fibrosis are not clear.In this study, we analyzed the effect of a lower level Ga–  As laser administration on muscle injury in an experi-mental rat model, trying to identify how oxidative stress,NF- k B activation, and iNOS overexepression, if present,were affected by LLLT therapy. Generally, a laser with awavelength of 600–984 nm is used in physical medicine,being a wavelength of 632.8 for a He–Ne laser and 904 nmfor a Ga–As laser the most frequent options in woundhealing. A Ga–As laser with a 904-nm wavelength waschosen in our study, because it is known that red light isoptimal for wound healing and Ga–As requires lowerdosage due to the high pulse power and high penetration. Woundhealinginvolvesformationofnewcapillariesandmigration of inflammatory cells (such as neutrophils andmacrophages) to the wound site and under this conditioncells of vascular srcin and inflammatory cells are a sourceof ROS [41] which can induce oxidative stress. In thepresentresearch,experimentalinjuryofthegastrocnemiusmuscle resulted in an inflammatory reaction that wasaccompanied by edema, angiogenesis, vasodilatation, andextracellular matrix formation. Moreover, the significantincrease in TBARS indicates the presence of ROS-inducedoxidativestressinthegroupofanimalswithmuscleinjury. We have recently reported a similar effect in an experi-mental model of Achilles tendon injury [15]. The increasedexpression of iNOS also indicated an excess generation of nitric oxide which by reaction with ROS to form peroxyni-trite could exacerbate the presence of oxidative andnitrosative stress [42].Oxidative stress is a potent pathogenic factor that leadsto various degenerative diseases, including tissue fibrosis.There is evidence that stimulation by ROS favors extra-cellular matrix deposition [43]. ROS have been related tofibrosis in different organs and tissues, such as liver, lung,kidney,orlens[18,20,44],andinallofthesefibrotictissues anabnormaldepositionofcollagenhasbeendescribed[45].Itisalsoknownthatoverexpressionoffibrogeniccytokinesand increased transcription synthesis of collagen can bedownregulated by the use of anti-oxidants [20,46]. More-over,aninversecorrelationbetweenlipidperoxidationand Fig. 3. Effects of trauma (T) and laser administration (TL) on collagen concentration. Valuesare means  SEM ( n ¼ 25–30). *  P < 0.05 against control.  #  P < 0.05 against trauma.Fig. 4. Effects of trauma (T) and laser administration (TL) on TBARS concentration. Valuesare means  SEM ( n ¼ 25–30). *  P < 0.05 against control.  #  P < 0.05 against trauma. 708 RIZZI ET AL.
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