ORIGINAL ARTICLE

Effects of 915 nm GaAs diode laser on mitochondriaof human dermal fibroblasts: analysis with confocal microscopy

Silvana Belletti & Jacopo Uggeri & Giovanni Mergoni &Paolo Vescovi & Elisabetta Merigo & Carlo Fornaini &Samir Nammour & Maddalena Manfredi & Rita Gatti

Received: 31 January 2014 /Accepted: 29 August 2014# Springer-Verlag London 2014

Abstract Low-level laser therapy (LLLT) is widely used intissue regeneration and pain therapy. Mitochondria are sup-posed to be one of the main cellular targets, due to thepresence of cytochrome C oxidase as photo-acceptor. Laserstimulation could influence mitochondria metabolism affect-ing mainly transmembrane mitochondrial potential (Δψm).The aim of our study is to evaluate “in vitro” the earlymitochondrial response after irradiation with a 915 GaAslaser. Since some evidences suggest that cellular response toLLLTcan be differently modulated by the mode of irradiation,we would like to evaluate whether there are changes in themitochondrial potential linked to the use of the laser treat-ments applied with continuous wave (CW) in respect to thoseapplied with pulsed wave (PW). In this study, we analyzedeffects of irradiation with a 915-nm GaAs diode laser onhuman dermal fibroblast. We compared effects of irradiationapplied with either CWor PWat different fluences 45-15-5 J/cm2 onΔψm. Laser scanning microscopy (LSM) was used in

living cells to detect ROS (reactive oxygen species) usingcalcein AM and real-time changes of and Δψm followingdistribution of the potentiometric probe tetramethylrhodaminemethyl ester (TMRM). At higher doses (45–15 J/cm2), fibro-blasts showed a dose-dependent decrement of Δψm in eitherthe modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed byany sign of toxicity, even if reactive oxygen species generationwas observed. At 5 J/cm2, CW irradiation determined a littledecrease (5 %) of the baseline level of Δψm, while oppositebehavior was shown when cells were irradiated with PW, witha 10 % increment. Our results suggest that different responsesobserved at cellular level with low doses of irradiation, couldbe at the basis of efficacy of LLLT in clinical application,performed with PW rather than CW modalities.

Keywords Bio-stimulation . CLSM . Laser . Low-level lasertherapy (LLLT)

Introduction

Low-level laser therapy (LLLT) is used to promote healing [1]and reduce pain and inflammation [2]. Many studies haveshown that wound healing process could be enhanced bytherapy supplied with helium neon (HeNe, 633 nm) or galliumarsenide (GaAs, 904 nm) lasers.

At cellular level, effects of low energy laser irradiationinclude cell proliferation, collagen synthesis, and release ofgrowth factors from cells [3]. Mitochondria seem to be themain targets of bio photo-stimulation with red and near-infra-red light [4]; some in vitro studies suggested that LLLTexposure could increase the activity of cytochromes C oxidase(unit IV of the mitochondrial respiratory chain), which is achromophore and absorbs light [5, 6]. Its effects concernedoxidative phosphorylation, energy metabolism, and ATP

S. Belletti : J. Uggeri : R. GattiUnit of Anatomy Histology and Embryology—Department ofBiomedical, Biotechnological and Translational Sciences(S.Bi.Bi.T), University of Parma, Parma, Italy

G. Mergoni : P. Vescovi : E. Merigo :C. Fornaini :M. ManfrediUnit of Oral Pathology, Medicine and Laser Surgery, Department ofBiomedical, Biotechnological and Translational Sciences(S.Bi.Bi.T), University of Parma, Parma, Italy

S. NammourDepartment of Dental Sciences, Faculty of Medicine, University ofLiège, Liège, Belgium

E. Merigo (*)Unit of Oral Pathology, Medicine and Laser Surgery, Department ofBiomedical, Biotechnological and Translational Sciences(S.Bi.Bi.T), University of Parma, via Gramsci, 14, 43126 Parma,Italye-mail: elisabetta.merigo@unipr.it

Lasers Med SciDOI 10.1007/s10103-014-1651-z

production [4–7]. Besides energetic metabolism, also the re-dox state of mitochondria is affected, with inherent changes ofredox-sensitive factors [8].

Mitochondria are organelles that have been classicallythought to be the power suppliers of the cell. Their role inthe cellular context has revealed several other contributions,including redox homeostasis, lipid modification, calcium ho-meostasis, and cell death processes. The inner membrane ofthe mitochondria maintains a transmembrane gradient of H+

known asmitochondrial potential, which drives mitochondrialATP synthesis [9].

Techniques of live cell for imaging mitochondria are con-tinually being improved to provide new information abouttheir morphofunctional features. Mitochondria are distributedthroughout the cytoplasm and exist in different sizes andshapes that range from small granular to highly filamentousshapes. Changes in mitochondrial morphology are strictlybound with changes in mitochondrial function [10].

Live cell imaging of mitochondria can be performed withspecific mitochondrial dyes that can be used to evaluate botht h e i r m o r p h o l o g i c a l a n d f u n c t i o n a l s t a t u s :tetramethylrhodamine methyl ester (TMRM) is a fluorescentlipophilic cationic dye commonly used to directly measuremitochondrial potential. More polarized mitochondria (i.e.,hyperpolarized, where the interior is more negative) will ac-cumulate more cationic dye, and depolarized mitochondria(interior is less negative) will accumulate less dye.

Combination of live stains with confocal microscopy hasyielded great new insights into the complexities of cells intheir living, non-fixed state. Mitochondria analysis can beperformed by recording the image data over time with detect-ing only fluorescence from the focal plane by rejecting theout-of-focus blur, thus performing optical sectioning.

Mitochondrial metabolism in response to laser irradiationhas been studied mostly with HeNe and GaAsAl lasers. Huet al. [11] showed an increment of ΔΨm after low powerintensity irradiation with HeNe laser (628 nm); on the con-trary, Wu et al. [12, 13], using CLSM, observed that a highfluence (120 J/cm2) low power (LPLI) irradiation with thesame laser caused a steep decrease in ΔΨm, mitochondrialpermeability transition (MPT), and apoptosis in ASTC cells.

Using fluorescence microscopy, Bortoletto et al. [14] andmore recently Pires Oliveira et al. [15] have observed somechanges of mitochondria activity and morphology after irra-diation with HeNe or 904 diode lasers.

The aim of our study is to evaluate early mitochon-drial response after irradiation at several fluences (45,15, 5 J/cm2) with a 915 GaAs laser, in a population ofdermal fibroblast cells. In particular, since some evi-dences [16] suggest that cellular response to LLLT canbe differently modulated by irradiation with pulsedwave light instead of continuous wave light, we aimedto compare treatments applied with continuous wave

(CW) with those applied with pulsed wave (PW) atthe same fluence.

Materials and methods

Laser irradiation

A GaAs diode laser device (LASEmaR 900™, Eufoton, Tri-este, Italy) with a wavelength of 915 nm was used in thisstudy. A laser beam delivered by an optical fiber of 0.32 mmin diameter was defocused at the tip of the fiber using concavelenses and was irradiated uniformly in a circular area, 1.24 cmin diameter. The irradiation was performed 10 mm above thecell layer. Laser light was tested in continuous and pulsedmode. In continuous mode, cells were irradiated with a poweroutput of 0.5W for 12, 36, and 108 s receiving 5, 15, and 45 J/cm2, respectively. In pulsed mode (pulse repetition rate of100 Hz and a pulse duration of 4 ms), cells were irradiatedwith a power output of 0.5W for 30, 90, and 270 s receiving 5,15, and 45 J/cm2, respectively.

Real power delivered by LASEmaR 900 was checked witha Powermeter (Ophir, Optronic, Israel) before each treatment.

Cell cultures

Primary culture of human dermal fibroblasts was derived fromskin biopsy of young health donor and routinely grown inDulbecco’s modified Eagle’s medium (DMEM) added with10 % fetal bovine serum (FBS), on plastic culture flasks(Costar Corporation, Cambridge, MA, USA). Cells weremaintained at 37 °C in a 5 % CO2 humidified incubator andsubcultured twice a week.

Confocal microscopy

For all experimental settings, 40.000 cells/cm2 were seededonto coverslips and after 24 h, the coverslip was placed into aspecial flow chamber [17] expressly designed for a real-timeprolonged observation of live cells with laser scanning mi-croscopy (LSM). The chamber was lodged in a commerciallyavailable incubation system (Kit Cell Observer, Carl Zeiss,Jena, Germany) that fitted the microscope stage and allowed acontinuous control of temperature and CO2. Samples weremaintained in this condition during the irradiation andthroughout image acquisition.

Observations were carried out on a LSM 510 Metaconfocal system scan integrated with the Axiovert 200Minverted microscope (Carl Zeiss, Jena, Germany) througha ×40/1.3 oil objective.

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Cell morphology and oxidative stress

Morphofunctional features of cell population after laser irra-diation were recorded in real time with LSM and flow cham-ber using calcein AM and propidium iodide (MolecularProbes) at the same time. Calcein AM is a vital probeemployed to detect changes during apoptosis [18]. Propidiumiodide (PI) is a non-vital probe for nucleic acid whose entryafter loss of membrane integrity allows to define the nuclearmorphology when calcein AM signal vanishes. Calcein wasexcited with the 488 laser line and the emission recordedthrough a 505–530-nm band pass filter. PI was excited withthe 543 laser line and the emission recorded through a 560long pass filter.

Calcein AM can be also used as real-time probe of intra-cellular oxidative activity, as an increase of its fluorescencesignal can be attributed to ROS generation [19].

Cells were loaded with calcein AM (2 µM) and propidiumiodide (2 µg) in culture medium, and a basal image wasacquired with LSM. Thus, cells were irradiated with theselected irradiation protocols and additional images werecollected.

Mitochondrial transmembrane potential (Δψ m)

Confocal microscopy allows a real-time visualization ofchanges in Δψm. Cell cultures prepared as above describedwere loaded with 50 nm tetramethylrhodamine methyl ester(TMRM, Invitrogen, Molecular probes, Eugene, OR, Usa).This probe [20] was rapidly accumulated into mitochondria ofliving cells driven by their Δψm providing a qualitative anal-ysis of its variation. Therefore, the intensity of its emission isproportional to the amplitude ofΔψm. This vital potentiomet-ric probe was excited with the 543 nm laser line and theemission recorded through a 560 long pass filter. After probeequilibration, images of a preselected field were acquired asbaseline and then irradiated. After the end of irradiation,sequential images of the same field were automatically ac-quired using the “Time series control,” performing a scansionevery 7.5 s. Acquired frames were processed for the semi-quantitative analysis of Δψm variation, using the softwaredeveloped by the microscope manufacturer. The softwarecalculated the average fluorescence intensity of each frame,and finally, the results were plotted as “percentage change” ofthe baseline fluorescence intensity versus time after laserirradiation.

Results

In this paper, we analyzed effects of 915-nmGaAs diode lasertreatment on cultures of human fibroblasts. Laser treatments

were exerted in the range of 5–45 J/cm2, the doses commonlyused with therapeutics aims.

Preliminarily, a live/dead test (calcein AM/propidium io-dide) excluded inherent toxic effects, even at the higherfluence employed (not shown), 24 h after laser treatment.

Besides qualitative data concerning morphology of livingcells, signal from calcein AM loaded cells is very sensitive toROS generation. Figure 1 shows calcein AM loaded cellsbefore and 5 min after the treatment with 45 J/cm2 in contin-uous modality. Fluorescent spots lighted up with a distributionrecalling mitochondria net (arrows). Higher fluorescent sig-nals were kept for only 5 min; after that, signal reverted tobasal level (not shown). Similar behavior of ROS generatedafter 45 J/cm2 laser treatment supplied with pulsed modalitywas comparable to those obtained with continuous modality.Treatments with 5 or 15 J/cm2 fluence did not induce signif-icant signal increase in either modalities employed.

Experiments were focused on mitochondrial transmem-brane potential (ΔΨm) that was studied incubating cells with50 nmTMRM. Fluorescent signal was analyzed with confocalmicroscopy in time-lapse modality. Before the treatment, cellswere loaded until probe equilibration: this represented thebasal image (T0) for each experiment. Then, cells were irra-diated either in continuous or pulsed mode with 45 or 15 or5 J/cm2 and time-lapse changes of TMRM fluorescence wererecorded for 600 s every 7.5 s. Fluorescent signal was mea-sured and expressed on graph as percentage of control values.

Figure 2 shows effects of treatment with 45 J/cm2 doseapplied either in continuous or in pulsed modality on TMRM-loaded cells. Data from time-lapse microscopy of the samemicroscopic field are shown on graph (Fig. 2). Both treat-ments caused a decrement of TMRM fluorescent signal. Incomparison with baseline levels, the amplitude was about15 % in pulsed mode and about 20 % in continuous. Initially,the observed decrements were very similar and with the sametime trend; then, beginning at about 100 s, TMRM fluores-cence showed an increment with relative parallel upslope ofboth modes (continuous and pulsed). Starting at about 200 s,the pulsed mode’s fluorescence exhibits a rapid incrementtoward base, whereas the continuous mode’s slowly increases,reaching basal level after over 500 s. Microscopic analysis offluorescent signal (Fig. 2B) showed that under basal condi-tion, fibroblasts loaded with TMRM had a heterogeneouslevel of fluorescence with the typical pattern of an evidentmitochondrial network and a negative nucleus (Fig. 2B).Figure 2B1) shows the same microscopic field 90 s afterirradiation at 45 J/cm2 applied in continuous modality.Figure 2B-B1 (the third panel) visualizes into the greenchannel the amplitude of difference obtained between Band B1 after software elaboration. Mitochondrial fluores-cence decrement concerned the population in heterogeneousmanner, more evident in some cells and in definite intracellu-lar areas.

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Also after irradiation at 15 J/cm2 (Fig. 3), TMRM signaldecreased with either the modalities of treatment employedwith behaviors very similar to those obtained for 45 J/cm2;even if amplitude of the decrease at 15 J/cm2 was lower than at45, signal from 15 J/cm2 pulsed treated cells returned at thebasal level more promptly.

Figure 4 shows images (upper and lower panels) and thetime-related fluorescence data (middle panel) comparing lasertreatment of 5 J/cm2 applied either in continuous (lower) or inpulsed modality (upper). After irradiation in continuous mode(empty circle), TMRM signal decreased slowly at about 95 %

of baseline in 120 s (empty arrow) and then returned to basalvalue. Fluorescence decrement between T0 (B) and 120-streatment (B1) is visualized in blue (Fig. 2(B-B1)).

Different and peculiar effect was recorded after irradi-ation in pulsed mode (filled circle). After 60 s (filledarrow), an overall increment of fluorescent signal wasevident. It peaked at about 10 % of the initial value andthen returned to basal value. In this experiment, the visu-alization of signal difference in the third panel (green)lights up an increase in fluorescence between basal leveland treated cells (Fig. 4(A1-A)).

Fig. 1 ROS generation after 45 J/cm2 CW. Real-time LSM imagesof the same microscopic fieldshowing ROS generation in fi-broblasts loaded withcalcein AM. a before and b aftertreatment with 45 J/cm2 applied inCW mode

Fig. 2 Real-time change ofΔΨm

after 45 J/cm2 treatment, CWversus PW. LSM images of thesame microscopic field,containing TMRM-loaded cells,before (B) and after 90 s of treat-ment with 45 J/cm2 (B1) appliedin CW mode. The third panel,visualized into blue channel, in-dicates the amplitude of differ-ence (B-B1) after software’s elab-oration. Data obtained from semi-quantitative analysis of all the ac-quired observations, either afterCWor PW treatment, are plottedin (a) as TMRM fluorescencepercentage change (see “Mate-rials and methods” for details)

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Discussion

Effects of 915-nm GaAs diode laser treatment on cultures ofhuman fibroblasts were analyzed with real-time confocal mi-croscopy in order to compare continuous or pulsed irradiationat different fluence (range 5–45 J/cm2).

Toxic effects were excluded even at the fluence of 45 J/cm2

applied with either continuous or pulsed modalities. Nonethe-less, when fibroblasts loaded with calcein AM were treatedwith this laser energy, transient fluorescent spots lighted upwith both the modalities used. This increase in fluorescentcalcein signal can be attributed to ROS generation [19] and isdescribed in numerous papers as an effect of laser treatmentsapplied with different modalities and energies. Several authorsdescribed that ROS generation by laser is usually linked to adecrease in ΔΨm [13]. Most of these analyzed effects oftreatments with high level energies and showed a cascade ofreactions that, starting from ROS, continued to mitochondrialpore transition and drove to cell apoptosis [12, 21].

Fig. 3 Real-time change of ΔΨm after 15 J/cm2 treatment, CW versusPW. Graphical analysis of fluorescence data obtained from TMRM-loaded cells after laser treatment with 15 J/cm2 applied with PW or CWmode. The relative fluorescence changes were calculated after semi-quantitative analysis of all acquired observations and performed withthe microscopy’s software (see “Materials and methods” for details)

Fig. 4 Real-time change ofΔΨm

after 5 J/cm2 treatment, CWversus PW. LSM images of thesame microscopic field,containing TMRM-loaded cells,before (A) and after (A1) treatmentwith 5 J/cm2 applied with PWandrelative visualization into thegreen channel (A1-A), showingthe amplitude of difference ob-tained from software’s elabora-tion. B represents the correspond-ing images of another microscop-ic field, obtained before (B) andafter (B1) treatment with 5 J/cm2

applied with CW. The semi-quantitative analysis (calculatedas described in “Materials andmethods”) of all fluorescence dataobtained from time-lapse obser-vations (PW vs CW) is plotted inthe middle panel graph. Thearrows indicate time points cor-responding to visualized imagesinA1 (full arrow) and inB1 (emptyarrow)

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In our experiments, real-time confocal microscopy wasused to follow effects of laser treatments on human dermalfibroblasts, loaded with TMRM that distributes proportionallyto transmembrane mitochondrial potential. Irradiation eitherin continuous or pulsed mode with 45 or 15 J/cm2 determinedmitochondrial depolarization: fluorescent signal decreaseswere proportional to energies used, and the amplitude washigher when treatment was applied with continuous than withpulsed modality. The decrease was transient, with a recoverytime dependent on the ways and on the energies applied: aftercontinuous laser, recovery lasted 600 or 400 s at the energiesof 45 or 15 J/cm2, respectively, while after pulsed treatment, itlasted 280 or 200 s. This observation suggests that the use ofhigher fluence independent from modality of administration(CW or PW) does not significantly change mitochondrialresponse of ΔΨm in our cell model.

Confocal microscopy and analysis of differences obtainedwith software elaboration prove to be very useful to followchanges occurring not only in specific cells, but also withpeculiar cellular localization. As visualized in panels reportingthe amplitude of difference obtained comparing images beforeand after treatment (images were built with software elabora-tion), fluorescence changes concerned the population in het-erogeneous manner, more evident in some cells and in definiteintracellular areas.

We can speculate that this behavior is linked to differentstatus of cells and of mitochondria: cells could be living indifferent phases of cell cycle and mitochondria could respondto different energy needs.

Most of in vitro studies about mechanism involving mito-chondria metabolism in cellular response to laser irradiationhave been performed with HeNe laser (628 nm). When thetreatment is applied with high fluence, ΔΨm decreases andMPT is triggered and is followed by apoptosis [13]. On thecontrary, some other papers report that when treatment isgiven at low power intensity, irradiation with HeNe laser(628 nm) at 0.5–2 J/cm2, an increment of ΔΨm in A2058melanoma cell line is induced [11].

When laser treatments were used at the fluence of 5 J/cm2,effects on mitochondrial potential showed discrepancies: afterirradiation in continuous mode, TMRM signal slightly de-creased and then returned to basal value, whereas after irradi-ation in pulsed mode, an increase in mitochondrial potentialwas shown (it peaked after 60 s).

Our experimental data indicates that, at this dose, themitochondrial metabolism can be differently influenced bythe delivering of light with pulsed mode. This mode extendsthe time required to reach total energy absorbed by cellulartarget (mitochondrial photo-acceptor cytochrome C), and thiscould mitigate the negative effects due to a high energy flowin a small time. Indeed, in continuous mode, at all dosestested, we have always observed a depolarization neverfollowed by toxicity, probably consistent with a transient

MPT opening that could operate in LLLT [22]. This phenom-enon has been recently linked to cyto-protective effects also inother cell models [23, 24]. The use of pulsed mode, usingenergy fractioned during time of irradiation, could be saferand also more effective in bio-stimulation because it uses arepetition pulse so that photons could be more efficientlyabsorbed by their cellular target.

Several studies have shown that LLLT performed eitherwith CWor PWcould exert an improvement in wound healingprocess in diabetic rats: with CW HeNe laser [25] and withHeNe or GaAs lasers [26] either in CWor PW.

Other comparative studies performed on in vitro cellmodels showed that 5 J/cm2 of energy developed by 660–670-nm LED light, if used with PW treatment, was moreefficient than CW in the same range of energy delivered [27,28].

For clinical applications, the use of pulsed light offers someadvantages. The most important is to generate less heating intarget tissues. Using this method, we can bring higher energiesin the deeper tissues, not damaging and potentially enhancethe effects.

Besides the advantage mentioned above, our results sug-gest that the different responses observed in fibroblasts with915-nm GaAs laser at the lower dose (relevant to LLLTtherapy), could help to explain the efficacy of LLLT in clinicalapplication, performed with PW rather than CW modalities.

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