Effect of IR laser on myoblasts: a proteomic study

Laser therapy is used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of this research was to get insight into possible benefits deriving from the application of an advanced IR laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of hypotrophic tissue. We studied the effect of IR laser treatment on proliferation, differentiation, cytoskeleton organization and global protein expression in C2C12 myoblasts. We found that laser treatment induced a decrease in the cell proliferation rate without affecting cell viability, while leading to cytoskeletal rearrangement and expression of the early differentiation marker MyoD. The differential proteome analysis revealed the up-regulation and/or modulation of many proteins known to be involved in cell cycle regulation, cytoskeleton organization and differentiation.

Introduction

Since the seventies laser therapy has been widely used in sports medicine, physiatry and rehabilitation to treat muscle diseases of different origins: myalgias, contusions, sprains, lacerations and damage due to heavy exercise. These diseases have in common the painful symptomatology, the inflammatory component and, in the case of injuries, the need to repair the tissue portion in which the muscle fibers have suffered damage. The application of laser therapy, either alone or combined with other treatments, both pharmacological and instrumental, has its rationale in the therapeutic effects that are attributed to laser radiation: analgesic, anti-inflammatory, anti-edema and ability to promote wound healing and tissue repair. A large amount of literature shows that laser radiation can affect the cell energy metabolism and ATP production, the response of immune cells to injury, the production of inflammation mediators, the behavior of
fibroblasts, and endothelial cells. Moreover, laser radiation can improve microcirculation, and relieve pain both indirectly through the effects mentioned above, and directly, by acting on receptors and nerve endings. 

Although many clinical studies give evidence for the effectiveness of IR laser therapy in the treatment of muscular disorders, thus justifying the wide application of laser treatments in clinical rehabilitation and sports medicine, in the literature there are conflicting results most likely caused by differences in the laser sources and treatment parameters that have been used. In the last few years significant progress has been made in understanding the mechanisms by which the IR laser therapy promotes the healing process and recovery of muscle tissue. Recent studies, carried out both in animal models and human subjects, demonstrated that pre-exercise treatment with IR laser can significantly delay muscle fatigue and accelerate post-exercise recovery. In rats, it has been found that laser treatment reduces the inflammatory response caused by experimentally induced muscle trauma and is able to block the effects of reactive oxygen species (ROS) release and the activation of NF-kB. Laser-induced changes in inflammatory biomarkers and significant muscle recovery have been observed also in a rat model of myopathy. Other authors found that, in traumatized muscle tissues, laser therapy induces an increase in activity of the complexes I, II, III and VI of the respiratory chain that may lead to an increase in ATP synthesis and faster muscle recovery. A study aimed at evaluating the effectiveness of IR laser radiation in promoting the recovery of atrophied skeletal muscles demonstrated that the laser treatment favours tissue repair through activation of satellite cells and induction of neoangiogenesis.

However, despite the fact that over the past decade numerous studies have significantly improved our knowledge, in many respects the effects of radiation emitted by different laser sources on muscle tissue and its repair mechanisms are far to be completely understood.

Paper

Fig. 1 MyoD expression in C2C12 cells after MLS laser treatment. a: control C2C12 cells. b: MLS treated C2C12 cells. c: quantitative expression of MyoD. Images obtained by immunofluorescence microscopy show that, in cells treated with MLS laser, MyoD expression increased and the transcription factor was mainly distributed in the nuclear and perinuclear area. Image analysis revealed that in treated cells MyoD increased by about 26%, in comparison with controls.

This paper reports the results of a study aimed to investigate the behavior of C2C12 cells exposed to the emission of a dual wavelength IR laser. The C2C12 skeletal muscle cell line has been derived from murine satellite cells and is widely accepted as a model to study the behavior of satellite cells, which play a crucial role in skeletal muscle regeneration and repair and are capable of repopulating damaged and atrophied muscle. The source chosen to perform the treatments was a synchronized IR dual-emission laser system, with wavelengths of 808 and 905 nm, respectively. Most of the recent studies reported the effectiveness of laser emissions in the range 800–830 nm as well as emissions around 905 nm in triggering a biological response in muscle tissue. Sources with multiple emissions are widely used in clinics but little is studied from the point of view of the
effects that induce in the cellular component of muscle tissue. We considered interesting to study the effects of a source that simultaneously emits two wavelengths which, on the basis of the literature, both can favour the recovery of homeostasis in diseased muscles. 

The behavior of C2C12 cells was assessed before and after exposure to laser treatments in terms of cell viability, morphology, proliferation, differentiation and proteomic profile.
To the best of our knowledge this is the first time that the effect of IR laser radiation on the proteomic profile of myoblasts is being studied.

Results and discussion

Effect of MLS laser treatment on viability, proliferation and differentiation of C2C12 cells

Murine myoblasts C2C12 were treated with a Multiwave Locked System laser (MLS laser) as described in the experimental section. Before proceeding to the appropriate morphological and proteomic studies, we analyzed the effect of the laser treatment on cell viability and proliferation. The trypan blue assay, performed both after a single exposure to the laser radiation and after 3 exposures carried out on consecutive days,
showed that in treated samples there were no significant changes in cell viability (over 98%) compared to the control. The cell count did not show significant differences after a
single exposure, whereas after 3 treatments with MLS laser, the cell number decreased moderately (about 23.4%) with respect to the control. Since MLS laser treatment does not affect cell viability, we hypothesized that the reduced proliferation rate of MLS laser treated cells was caused by the triggering of a differentiative process. To verify our hypothesis we analyzed expression and distribution of MyoD, which is widely recognized as an early marker of myoblast differentiation. In fact it is known that in skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. In treated cells MyoD increased by about 26%, in comparison with controls (Fig. 1c), and was mainly distributed in the nuclear and perinuclear area (Fig. 1a and b). The increase in MyoD expression and the morphological changes in the cytoskeleton structure observed in treated cells strongly suggest that MLS laser radiation triggered a differentiation process in C2C12 cells. 

Effect of MLS laser treatment on cytoskeleton organization 

The organization of the cytoskeleton network is a crucial factor in determining cell shape, regulating cell adhesion/migration, transducing signals and triggering intra and extracellular pathways. Therefore, the three major cytoskeleton components, i.e. actin microfilaments, microtubules and intermediate filaments were studied by immunofluorescence microscopy analysis. Control C2C12 cells showed the expected actin distribution (Fig. 2a): high expression in the perinuclear area, a clearly distinguishable ‘‘actin ring’’ close to the plasma membrane (arrow) and some stress fibers. After a cycle of three laser treatments the ‘‘actin ring’’ delimiting each individual cell
disappeared. The cells tended to align and fuse to form tubes, filaments running parallel to the axis of the tubes appeared (arrows), the perinuclear area with high actin expression was thicker (Fig. 2b). The specific staining for tubulin revealed, in control cells, a radial distribution of the microtubules starting from the organization centre close to the nucleus (Fig. 2c). In treated samples a redistribution of the microtubules was observed: they were oriented parallel to the major axis of the cells and passed from one cell to another without interruption (arrows) (Fig. 2d). The intermediate filaments were studied by immunofluorescence staining of vimentin, which is their major constituent. The analysis of protein expression and distribution in control cells showed that the network of filaments was more dense in the perinuclear area, where it had the shape of a ball
(Fig. 2e). In the cells treated with MLS laser, the intermediate filaments were parallel to the longitudinal axis of the cells (arrows), which appeared elongated and aligned to form tube-like structures (Fig. 2f). In summary, C2C12 cells, subjected to MLS laser treatment,
showed elongated shapes and we realigned and fused to form structures with two or more nuclei among which were no longer recognizable interposed membranes (tubes). These cytoarchitectural changes support the occurrence of a differentiation process since the formation of a longitudinal microtubule array is an early event in myogenic differentiation. It is also known that the reorganization of intermediate filaments and shifts from one to the other of the two major components, vimentin and desmin, occur during myogenesis. Remodelling of actin microfilaments with the formation of stress fiber like structures has a very important role leading to myofibrillogenesis and is regulated by actin binding proteins and phospholipase D.

Molecular BioSystems

Fig. 2 Cytoskeleton organization in C2C12 cells after MLS laser treatment. a, b: actin immunostaining. c, d: tubulin immunostaining. e, f: vimentin immunostaining. In control C2C12 cells (a) actin resulted highly expressed in the perinuclear area and formed a distinguishable ‘‘actin ring’’ close to the plasma membrane (arrows). Some stress fibers were present. After a cycle of three laser treatments (b) the ‘‘actin ring’’ delimiting each individual cell disappeared. The cells tended to align and fuse to form tubes, filaments running parallel to the axis of the tubes appeared (arrows), the perinuclear area with high actin expression was thicker. In control cells (c), microtubules showed a radial distribution starting from the organization centre close to the nucleus. In treated samples (d) the microtubules oriented parallel to the major axis of the cells and passed from one cell to another without interruption (arrows). In control cells (e), the network of intermediate filaments was more dense in the perinuclear area, where it had the shape of a ball. In laser treated cells (f), the intermediate filaments were parallel to the longitudinal axis of the cells (arrows), which appeared elongated and aligned to form tube-like structures.

Effect of MLS laser treatment on the proteome of C2C12 cells

In an attempt to identify the molecular changes induced by laser treatment of C2C12 cells, we have studied the protein expression pattern before and after laser treatment using 2-DE based proteomics. To ascertain the reproducibility of results 2-DE was performed three times for each protein sample. Fig. 3 shows a representative gel image. Image analysis using Progenesis Same Spot software allowed us to identify about 120 significantly (Anova p value, p ≤ 0.05) and consistently up- or down-regulated protein spots with fold changes greater than 1.5 in terms of average normalised volumes in both triplicate gels of two independent experiments. Of these spots, 89 were up-regulated
and 32 down-regulated in comparison to the control cells. Fig. 3 shows the significantly regulated spots, identified by MALDITof mass spectrometry.

About eighty spots, corresponding to major protein variations, were cut from gels, destained, digested with trypsin and subjected to peptide mass fingerprinting followed by database searching. MALDI-TOF MS analysis allowed the unambiguous protein identification of 52 protein variations, corresponding to 42 proteins. Table 1 summarizes all the information obtained by protein identification. Protein numbering corresponds to
that shown in Fig. 3. Swiss-Prot accession number and protein name are also included. The comparison between theoretical and measured molecular weight and pI values contributes to confirm the MASCOT search results in most cases. The MASCOT search results are reported in Table 1, showing experimentally measured peptide masses matching the theoretical ones from Swiss-Prot/UniProt entries, the percentage of the protein sequence covered by the matching peptides (sequence coverage),
and the probabilistic score.

The identification of some protein spots, both selected as up-regulated proteins (spot No. 7, 13–15, 17, 40) and down-regulated ones (spot No. 4, 6, 37, 44), resulted in the same
proteins: vimentin (spot No. 4, 13–15), actin γ (or β) (spot No. 17 and 44), tropomyosin α-3 chain (spot No. 6 and 40) and Rab GDP dissociation inhibitor β (spot No. 7 and 37). The position of these protein spots is clearly different. Fig. 4 shows details of the two-dimensional reference maps. Both tropomyosin α-3 chain and Rab GDP dissociation inhibitor β are associated with two spots with equal molecular weight but different isoelectric points. The observed shift in positions of these proteins could represent splice variants and/or post-translational modifications (i.e. phosphorylation) rather than an increase or decrease in the absolute amounts. Vimentin and actin γ (or β), identified
by the analysis of protein spots up-regulated following laser treatment, have an apparent molecular weight higher than the theoretical one. This phenomenon was also observed for other proteins identified by the analysis of up-regulated protein spots. As we can note from Fig. 3 and Table 1, desmin (spot No. 16), pyruvate kinase isozymes M1/M2 (spot No. 22) and elongation factor 2 (spot No. 21) show a measured molecular weight higher than the theoretical one. We hypothesize that MLS laser treatment could induce the stabilization of pre-existing covalent polymeric species or their formation. 

Classification and functional analysis of modulated proteins 

In order to gain insight into the biological significance of the proteins identified by proteomic analysis, the 42 differentially expressed proteins, identified by MALDI-Tof peptide mass finger printing of 52 isolated protein spots, were categorized according to the DAVID bioinformatics tool. Concerning biological processes, the identified proteins were distributed into categories; we report here the results obtained with the PANTHER (Protein Analysis Through Evolutionary relationships) classification system (Table 2). The main biological process in which the identified proteins were involved was ‘‘protein metabolism and modification’’ (38.1%), followed by ‘‘cell structure and motility’’ (28.6%),
‘‘carbohydrate metabolism’’ (11.9%), and ‘‘induction of apoptosis’’ (7.1%). The four tropomyosin isoforms identified and assigned to the ‘‘cell structure and motility’’ class can be categorized also in the ‘‘muscle contraction and development’’ class (not shown). Similarly heat shock proteins can be classified also as ‘‘stress response proteins’’. It is well known that the main function of the heat shock proteins is to provide thermotolerance and cytoprotection. Hsp-β1, up-regulated after MLS laser treatment, belongs to the small heat shock protein group. It is overexpressed during different stages of cell differentiation
and development and it is thought to have an essential role in the differentiation processes of numerous tissues. 

The membership of most of the identified proteins to the class ‘‘protein metabolism and modification’’ clearly indicates a cellular response toward specific anabolic events, probably related to cytoskeleton network remodelling, leading to the morphological changes observed and described above. It is also remarkable that about 30% of the changes shown by proteomic analysis in laser treated samples concern proteins involved in cell structure and motility, such as actin γ/β, tropomyosin α and β chains, vimentin, desmin, LIM domain and actin-binding protein, fascin, cofilin-1, many of which are actin-binding proteins and/or have been found to have a role in myogenesis.
It has been demonstrated that the LIM domain and actin binding protein increases when myoblasts are induced to differentiate and then progressively declines in myotubes.
Therefore, the increase in the LIM domain and actin-binding protein we observed fits with a scenario of differentiation at the early stages. Another interesting identified protein is
a-enolase that results up-regulated after MLS laser treatment. Although it is clusterized into the class ‘‘carbohydrate metabolism’’, it is known that α-enolase is also involved in other cellular processes. It has been recently demonstrated that enolase isoforms interact with microtubules during muscle satellite cell differentiation contributing to the regulation of the cytoskeletal filaments dynamism that occurs during the transition
from myoblasts to myotubes. NLR family pyrin domain-containing protein 10 (NLRP 10), heterogeneous nuclear ribonucleoprotein K (HNRNP K) and galectin-3 following the PANTHER classification system were assigned to the ‘‘induction of apoptosis’’ class but they are also involved in other important processes.

Fig. 3 Representative reference 2-DE gel of C2C12 cells. Cell lysates of control C2C12 cells and MLS-treated C2C12 cells were resolved by 2-DE. IEF was carried out on nonlinear wide-range IPGs (pH 3–10; 18 cm IPG strips) and achieved using the Ettant IPGphort
system. Sample load, 800mg per strip, was successively performed by cup loading in the IPGphor Cup Loading Strip Holders. The second dimension was carried out on 9–16% polyacrylamide linear gradient gels (18 cm 20 cm 1.5 mm). Protein spots were visualized by colloidal coomassie blue staining. 2-DE gels were analysed using the Progenesis SameSpot software package. The arrows point to differential protein spots identified with the peptide-mass fingerprinting

NLRP 10 is one of 14 pyrin domain containing members of the NOD-like receptor family of cytoplasmic receptors. It is an intracellular protein involved not only in apoptosis but also in the immune system function. In fact it is believed that NLRP 10 helps to regulate the inflammatory response. NLRP 10 reduces inflammatory and innate immune responses by inhibiting the activity of two proteins associated with the inflammasome:
caspase-1 and PYCARD. Although the increase in NLRP 10 found after laser treatment does not seem to be connected to differentiation, it could represent one of the mechanisms underlying the anti-inflammatory effect attributed to the laser therapy.

Fig. 4 Protein shift position of actin γ (or β), vimentin, tropomyosin α-3 chain, and Rab GDP dissociation inhibitor β upon MLS-treatment of C2C12 cells. The panels show the regions, selected by representative 2-DE gels, with actin γ (or β), vimentin, tropomyosin α-3 chain, and Rab GDP dissociation inhibitor β localization. The indicated proteins shift their position in response to MLS-treatment due, likely, to post-translational modification.

HNRNP K belongs to the subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs). These proteins are associated with pre-mRNAs in cell nucleus and are known to
influence pre-mRNA processing and other aspects of mRNA metabolism and transport. Experiments on animal models showed that HNRNP K is required for axonogenesis during development and several of its RNA targets are under strong post-transcriptional control during the regeneration process.

The increase in HNRNP K observed in laser treated cells could be an intriguing starting point for future research, since it has been shown that IR laser therapy promotes the regeneration of nerve fibers. HNRNP K is also thought to have a role in cell cycle progression, therefore the increase in expression could also be connected with the beginning of a differentiation process.

Galectin-3 plays a key role in several intracellular and extracellular processes. Documented intracellular functions are the regulation of cell growth, apoptosis and cell cycle. Extracellular function consists in mediating/modulating cell to extracellular matrix adhesive interactions. Recent studies indicate galectin-3 as a mediator of signal transduction events on the cell surface as well as a mediator of a variety of extracellular processes such as angiogenesis, neuronal functions, endocytosis and possibly exocytosis.
Finally, we would like to highlight the identification of PP1 as one of the up-regulated proteins in MLS laser treated cells.

Interestingly the PP1 catalytic subunit protein is included in each of the three main classes. PP1 is a major eukaryotic protein serine/threonine phosphatase that regulates an enormous variety of cellular functions through specific associations with regulatory
subunits. PP1, primarily known for its role in the carbohydrate metabolism, actually regulates functions such as actin and actomyosin reorganization, cell shape and cell adhesion, muscle contraction/relaxation.
To assess the identity of PP1 we performed an immunoblot analysis using a specific anti-PP1 antibody. Actin and enolase were selected to undergo a confirmatory test as
well. Fig. 5 shows immunoblot results in comparison with the colloidal coomassie staining. Western blot analysis also confirms the up-regulation of enolase and the up-regulation of PP1 in laser treated cells, according to 2-DE gel image
analysis.

Another interesting aspect highlighted by the DAVID classification system as a function of the keywords is that 81% of the identified proteins are classified as ‘‘phosphoproteins’’
(Table 3). Some of these proteins actually are PP1 substrates or interact with it (i.e. cofilin, heterogeneous nuclear ribonucleoprotein K, enolase, heat shock proteins, peptidylprolyl isomerase, vimentin).

The overexpression of cofilin is notable. Cofilin is a protein associated with the cytoskeleton that binds actin and reversibly controls polymerization and depolymerization in a pH-sensitive manner; the ability of cofilin to control actin polymerization is known to be regulated by reversible phosphorylation. Cofilin
is phosphorylated by LIM kinase 1, which abolishes its ability to de-polymerize actin, and dephosphorylated by PP1 and PP2A.

PP1 and PP2A play an important role in myoblast differentiation. It has been shown that inhibition of PP1 and PP2A by okadaic acid blocks myogenesis by altering the MyoD binding activity and depletion of PP1 abolishes the ability of myoblasts to differentiate into myotubes. Proteomic analysis of samples exposed to MLS laser treatments for 3 consecutive days showed an increase in PP1 but no significant changes in PP2A. This finding completely agrees with the results of other authors, who examined PP1 and PP2A activities during various stages of myogenesis in rat skeletal muscle cells. PP1 activity increased progressively in cultures from 2 to 5 days, PP2A activities remained constant in days 2–4 cultures and increased sharply on day 5. An indirect proof of the key role played by PP1 in muscle homeostasis is the decrease of PP1 levels, associated with a decrease in metabolic enzymes, observed in hypotrophic and sarcopenic muscles.

Finally it is also relevant that 28.7% of the identified proteins are ‘‘ATP-binding proteins’’, 19% are ‘‘coiled coil proteins’’ and 16.7% ‘‘actin-binding proteins’’. The increased availability of ATP induced by exposure to the red-IR laser radiation could be correlated with the net increase of proteins capable of binding ATP. Changes in the expression of actinbinding proteins, as well as those in typical intermediate filament proteins (here classified in part as ‘‘coiled coil’’ proteins), are events related to the general cytoskeletal rearrangement also observed by fluorescence microscopy (Fig. 2).

Fig. 5 Validation of the PP1 identity by immunoblot analysis. 100 μg of protein extracts from control and MLS treated C2C12 cells were separated by 2-DE and transferred on a PVDF membrane. The blots were incubated with anti-PP1 antibody. Anti-actin and anti-enolase antibodies were used as loading and position control.
Blots were visualized by autoradiography (c, d). The panels represent the 2-DE selected regions in which localize PP1, actin and enolase. The upper panels (a, b) show the colloidal coomassie blue stained gels

MLS laser treatment induces a significative PPPs activity increase

The identification of PP1 as one of the up-regulated proteins in response to MLS laser treatment and the observation that 81% of the identified proteins are proteins whose function depends on the phosphorylation status led us to investigate whether there were
changes in the total cellular protein serine/threonine phosphatases activity. PPPs activity assays, performed on cell lysates obtained after MLS laser treatment of C2C12 cells, show a 1.8 fold increase with respect to untreated cells (Fig. 6) confirming the importance of
specific phosphorylation/dephosphorylation events in maintaining the integrity of intermediate filaments and other fundamental biological functions. Fig. 6 shows the results obtained for other enzymes: lactate dehydrogenase, enolase and pyruvate kinase. All these proteins are overexpressed after MLS laser treatment and show an increase in their enzymatic activity

Fig. 6 Enzymatic assays of some overexpressed proteins and cellular protein phosphatases. The enzymatic activity of protein serine/threonine phosphatases (PPPs), lactate dehydrogenase (LDH), enolase, and pyruvate kinase (PK) was determined in cell lysates by specific tests. The figure shows the activity fold increase observed in MLS-treated C2C12 cells with respect to control cells. Results shown represent means of two experiments in duplicate (SEM (*p r 0.05)).

Experimental

Cell culture

Murine myoblasts (C2C12 skeletal muscle cell line) were routinely cultured in growing medium consisting of Dulbecco’s Modified Eagle’s Medium supplemented with 100 μg ml-1 streptomycin, 100 U ml-1 penicillin, 2 mM glutamine and 10% fetal bovine
serum (FBS). Cells were incubated at 37 1C and 5% CO2. All the reagents for cell culture were purchased from Sigma Chemical Co. (St Louis, MO, USA).

MLS laser treatment

The treatments have been performed with a Multiwave Locked System (MLS) laser (ASA Srl, Vicenza, Italy), a device already used for some years in clinics (FDA approved and CE certified instrument) and specifically applied in physical medicine and pain therapy. It is a high power (average power up to 1.1 W, class IV) IR laser with two synchronized sources (laser diodes).

The two modules have different wavelengths, peak power and emission mode. The first one is a pulsed laser diode, emitting at 905 nm, with a peak optical power = 25 W; each pulse is composed of a pulse train (single pulse width = 100 ns, maximum frequency 90 kHz), thus varying the average power delivered to the tissue. The frequency of the pulse trains may be varied in the range 1–2000 Hz. The second laser diode (808 nm) operates in continuous mode (power 1.1 W) or in pulsed mode (pulses repetition rate 1–2000 Hz), mean optical power output = 550 mW, duty ratio 50% independent of the pulse repetition rate. The two propagation axes are coincident. 

For the treatment, cells were seeded in the central 8 wells of a 24-multiwall plate. The plate was placed inside a plexiglass support, specifically designed and built. On the top of the support there was a central groove in which the laser handpiece slided. The plate was perfectly aligned with the handpiece, at a distance of 3 cm from it, so that the spot formed by the two superimposed laser beams had a diameter equal to that of a single well (13 mm). The support allowed us to perform a homogeneous scan of 8 samples at the same time, by moving the spot at a constant horizontal velocity above the 8 treated wells (5.6 cm s1: each scan of 8 wells lasted 20 s), in order to have the same radiant energy impinging into each well (B68 J for the whole treatment). Treatment parameters were 1500 Hz frequency and 8 min total scan time. The scan mode is also extensively used in clinics because it allows us to treat easily larger areas and, together with the other treatment parameters chosen, contributes to achieve the desired effects avoiding any side effects.

The treatment was repeated once a day, for 3 consecutive days under sterile conditions. The treated samples were compared with controls maintained under the same conditions, except for the laser exposure.

Viability and proliferation

Cell viability was assessed by the Trypan Blue assay after a single exposure and 3 exposures (once a day for 3 consecutive days) to MLS treatment. The treated samples were compared to untreated controls. After the MLS treatment, the samples were washed and cell detachment was obtained by treating with trypsin–EDTA for 3–4 minutes. Then the cells were centrifuged and resuspended in a solution of phosphate buffered saline (PBS) and Trypan Blue (dilution factor: 2). The dye is able to penetrate selectively into dead cells. After 5 min of incubation, cell counts were performed by using a Neubauer haemocytometer.

Immunofluorescence analysis

At the end of the experiments, cells were fixed for 5 min in cold acetone, then washed in phosphate buffered saline (PBS). After blocking unspecific binding with PBS containing 3% bovine serum albumin, cells were incubated overnight at 4 1C with the specific anti-MyoD (Santa Cruz Biotechnology, SC-32758), anti-a-actin (Millipore, MAB1501X), anti-tubulin (Upstate Biotechnology, 05829) and anti-vimentin (Chemicon, MAB1681) antibodies. The cells were then incubated with the fluorescein isothiocyanate (FITC) conjugated specific secondary antibodies (specifically: anti-mouse IgG (Chemicon Int, AP 124-T) for antitubulin antibody and anti-mouse IgM (Chemicon Int, AP 132-T) for anti-vimentin antibody). Cells incubated with anti-α actin antibody did not need incubation with the secondary antibody
since a mouse anti-actin Alexa Fluors 488 conjugated was used.

Negative controls were obtained by omitting the primary antibodies. Samples were evaluated using an epifluorescence microscope (Nikon, Florence, Italy) at 100 magnification and imaged by a HiRes IV digital CCD camera (DTA, Pisa, Italy).
Image analysis was performed by extracting, for each cell image, the region of interest (ROI) by appropriate software (Image Pro Plus). Then, the mean pixel value (16 bit, gray level) related to the mean fluorescence intensity and therefore to the specific epitope detection was calculated.

Data analysis

Three different experiments were carried out in triplicate. For viability and proliferation assays at least 10 counts per sample were carried out and the mean value was calculated.

For immunofluorescence analysis, at least 30 cells per slide were scored in 10 random fields per slide, and the data were expressed as mean SD. Statistical significance was determined using a Student’s t-test. A p value lower than 0.05 was considered statistically significant.

Proteomic sample preparation and 2-DE

For 2-DE, MLS treated and control cells were harvested by centrifugation at room temperature. The pellet was washed twice in water and resuspended in 8 M urea, 4% CHAPS, and 10 mM DTT. After sonicating briefly, protein extracts were clarified by centrifugation at 14 000 g for 10 min. The protein concentration of each purified sample was determined using the 2D Quant kit (GE Healthcare, USA). For each experimental
condition 2-DE replicate gels (n = 3) were made using independent experiments, in order to assess biological and analytical variations.
IEF (first dimension) was carried out on nonlinear wide-range IPGs (pH 3–10; 18 cm long IPG strips; GE Healthcare, Uppsala, Sweden) and achieved using the Ettant IPGphort system (GE Healthcare).

IPG-strips were rehydrated with 350 ml of lysis buffer and 2% v/v carrier ampholyte, for 12 h at room temperature. Sample load, 800  μg per strip, was successively performed by cup loading in the IPGphor Cup Loading Strip Holders (GE Healthcare), with the sample cup system at the anodic side of IPG strips. IEF was then achieved according to the following voltage steps, at 20 1C: 30 V for 30 min, 200 V for 2 h, 500 V for 2 h, from 500 to 3500 V for 30 min, 3500 V for 5 h, from 3500 to 5000 V for 30 min, 5000 V for 4 h, from
5000 to 8000 V for 30 min, 8000 V until a total of 95 000 V h 1 was reached. After focusing, prior to the second-dimension separation, IPG strips were equilibrated in equilibration buffer (6 M urea, 75 mM Tris–HCl pH 8.8, 29.3% glycerol, 2% SDS) containing 1% (w/v) DTT for 15 min and then in the same equilibration buffer containing 2.5% iodoacetamide for a further 15 min.
The second dimension separation was carried out on 9–16% polyacrylamide linear gradient gels (18 cm 20 cm 1.5 mm) at 40 mA per gel constant current and 10 1C until the dye front reached the bottom of the gel. Protein spots were visualized by colloidal coomassie blue staining. The stained gels were scanned with the Epson Expression 1680 Pro image scanner.

Image analysis

Scanned images (16-bit grayscale) were processed and statistically evaluated with Progenesis SameSpots software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Both manual and automatic alignment was used to align the images. A control group and a ‘‘laser treated’’ group containing three technical replicates were created and only spots present in all the replicates were taken into consideration for subsequent analysis. The two groups were compared with each other and fold values as well as p-values of all spots were computed by the above-mentioned software using one way ANOVA analysis. All spots were prefiltered and manually checked before applying the statistical criteria (Anova p ≤ 0.05 and fold ≥ 1.5). Normalized spot volumes, instead of spot intensity, were used in statistical processing. Protein identification involved only spots that fulfilled the statistical criteria. Experimental pI and Mw value were estimated using MW protein markers and some identified proteins selected as markers.

In-gel digestion and MALDI-TOF analysis

Protein spots were manually excised from gels and each sample was transferred to a 1.5 ml Eppendorf tube, washed twice in 50 mM ammonium bicarbonate (NH4HCO3)/CH3CN 1/1 for 15 min and then de-hydrated in CH3CN. Dried samples were re-swelled in NH4HCO3 containing 10 mM DTT (freshly prepared) and incubated for 30 min at 56 1C; the excess liquid was then removed and replaced with the same volume of
freshly prepared 55 mM IAA in 25 mM NH4HCO3. After 30 min of incubation at room temperature in the dark, the gel particles were washed twice with NH4HCO3/CH3CN 1/1 for 15 min, de-hydrated in CH3CN and dried in a vacuum centrifuge. Each sample was incubated for 1 h at 37 1C in 20  μl of 20  μg ml1 trypsin solution (Trypsin Proteomics Sequencing Grade T6567, SIGMA) in 40 mM NH4HCO3 with 10% CH3CN.

An additional 30  μl of 40 mM NH4HCO3 with 10% CH3CN were added to each sample and incubated overnight at 37 1C. The reaction was stopped by adding a final concentration of 0.1% trifluoroacetic acid. The supernatant was collected and the gel
was further extracted with 0.1% trifluoroacetic acid in 50% CH3CN.79,80 The extracts were combined and then analysed on a MALDI-TOF/TOF mass spectrometer Ultraflex III (Bruker Daltonics, Bremen, Germany) by using Flex Control 3.0 as data acquisition software. A 0.75  μl volume of the sample was mixed with 0.75  μl of the matrix (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% (v/v) CH3CN and 0.5% (v/v) TFA)
on the anchorchip target plate and allowed to dry. Spectra were acquired in the reflectron mode over the m/z range 860–4000 for a total of 500 shots. The instrumental parameters were chosen by setting the ion source 1 at 25 kV, ion source 2 at 21.5 kV, the pulsed ion extraction at 20 ns and the detector gain at 7.7x.

The instrument was externally calibrated prior to analysis using the Bruker Peptide Calibration standard kit. All the resulting mass lists were cleaned up from eventually present contaminant masses, such as those from matrix, autodigestion of trypsin and keratins. Mass fingerprinting searching was carried out in Swiss-Prot/TrEMBL databases using MASCOT (Matrix Science Ltd., London, UK, http://www.matrixscience.com)
software. The taxonomy was restricted to Mus musculus, a mass tolerance of 50 ppm was allowed, and the number of accepted missed cleavage sites was set to one. Alkylation of cysteine by carbamidomethylation was assumed as fixed modification. The experimental mass values were monoisotopic. No restrictions on protein molecular weight and pI were applied. The criteria used to accept identifications included the extent of sequence
coverage, number of matched peptides and probabilistic score sorted by the software.

Western blot

Western blot analysis was performed to validate the identity and the differential expression of PP1, enolase and actin. 100 mg of protein extracts from control and MLS treated C2C12 cells were separated by 2-DE as previously described and transferred onto a PVDF membrane (Millipore). The blots were incubated with anti-actin (Santa Cruz Biotechnology, SC-1615), antienolase (Santa Cruz Biotechnology, SC-7455) and anti-PP1
antibodies (Santa Cruz Biotechnology, SC-7482) in blocking buffer (PBS, 2% nonfat dry milk, 0.1% v/v Tween-20). After incubation with secondary antibodies, the blotting was developed by using the ECL plus immunodetection system ECL (GE Healthcare) and visualized by autoradiography.

Cluster analysis

The differentially expressed proteins were subjected to functional pathway analysis using DAVID database (http://david.abcc.ncifcrf.gov/home.jsp) for better understanding of the biological context of the identified proteins and their participation in various physiological processes. UniProt accession numbers of the 42 differentially expressed proteins identified in our study were uploaded and mapped against the Mus musculus reference dataset to extract and summarize functional annotation associated with individual or group of genes/proteins and to identify gene ontology terms, molecular function, biological process and important pathways for each dataset.

Determination of protein serine phosphatases, pyruvate kinase, enolase and lactate dehydrogenase activities 

Protein Serine Phosphatases (PPPs) activity was determined in C2C12 cell lysates from three independent experiments. MLS-treated and not treated C2C12 cells were quickly rinsed in ice-cold phosphate-buffered saline (PBS, 10 mM sodium phosphate and 0.15 M NaCl, pH 7.2), and freezed. After thawing the material at room temperature, the lysis was performed at 4 1C in 50 mM Tris, pH 7.4, containing 5 mM dithiothreitol and Sigma protease inhibitors mix (1/100, v/v). After 30 min of incubation on ice, lysates were sonicated (three short bursts) and centrifuged at 12 000g in a microcentrifuge at 4 1C for 30 min. Supernatants were quantified with respect to proteins content by the Bradford method. PPPs activity was determined using p-nitrophenyl phosphate as the substrate. All enzymatic activity tests were performed in duplicate. The substrate (4 mM) was dissolved in 25 mM Tris–HCl buffer, pH 7.2, containing 5 mM dithiothreitol, 20 mM sodium–potassium DL-tartrate and 0.1 mM sodium orthovanadate. Tartrate and orthovanadate were added in order to inhibit protein tyrosine phosphatases, lysosomal acid phosphatases and non-specific phosphatases.

The reaction was stopped with 0.1 M KOH and the released p-nitrophenolate ion was measured by reading the absorbance at 400 nm (e = 18 000 Ml cm1). The activity measured under these conditions was completely inhibited by 0.01 mM cantharidic acid,
a specific and strong inhibitor of all PPPs. Statistical significance was determined using a Student’s t-test. A p value lower than 0.05 was considered statistically significant.
Pyruvate kinase (PK) activity was determined at 30 1C according to Bergmeyer, with slight modifications, continuously following the NADH oxidation at 340 nm, using an UV-2100 spectrophotometer (Shimadzu, Columbia, MD). The assay mixture contained in 1 ml final volume consisted of 50 mM triethanolamine (pH 7.6), 8 mM MgSO4, 5 mM EDTA, 75 mM KCl, 1.5 mM ADP, 0.15 mM NADH, 60 units of lactate dehydrogenase. The reaction was started by adding substrate (0.8 mM phosphoenolpyruvate). Enolase activity was determined at 30 1C according to Bergmeyer, with slight modifications, continuously
following the NADH oxidation at 340 nm, using an UV-2100 spectrophotometer (Shimadzu, Columbia, MD). The assay mixture contained in 1 ml final volume consisted of 80 mM triethanolamine (pH 7.6), 3.3 mM MgSO4, 1.1 mM ADP, 0.2 mM NADH, 20 units of
lactate dehydrogenase, 3 units of pyruvate kinase. The reaction was started by adding substrate (0.9 mM phosphoglycerate). Lactate dehydrogenase (LDH) activity was determined at 30 1C according to Bergmeyer, with slight modifications, continuously following the decrease of NADH at 340 nm, using an UV-2100 spectrophotometer (Shimadzu, Columbia, MD). The assay mixture, contained in 1 ml final volume, consisted of 100 mM phosphate buffer pH 7.0 and 0.2 mM NADH. The reaction was started by adding substrate (0.77 mM pyruvate). The value of 6.22 mM1 cm1 is considered to be the NADH molar extinction coefficient. One unit of activity is defined as that quantity of enzyme which transforms one mmole of substrate in one minute at 30 1C.

Conclusions

The aim of this study was to investigate the response of myoblasts to IR laser treatment in order to get further insights into the cellular and molecular mechanisms underlying the
effects of laser therapy on muscle tissue described in clinical studies and on animal models. Our results show that laser treatment, with the source and parameters chosen, did not affect cell viability but induced a decrease in cell proliferation and increase in expression of the early differentiation marker MyoD, associated with changes of cell morphology and cytoskeletal architecture leading to the formation of tube-like structures. Taken together, these findings suggest that the exposure to IR laser triggers a differentiation process in myoblasts.

The analysis of differential expression in the proteomic profile of laser treated and untreated cells, which to the best of our knowledge had never been performed before, further confirmed in treated cells a scenario of differentiation process in its early stages. In fact, following laser exposure, numerous proteins known to be involved in cell cycle regulation, cytoskeleton organization and differentiation showed a significant increase or
modulation. The fact that IR laser treatment seems to be able to promote myoblast differentiation in vitro could in part explain the regenerative and reparative effects attributed to laser therapy when applied to muscle injury in clinics. Very interestingly, the proteomic analysis also revealed the increase of numerous ATP-binding proteins and proteins involved in the regulation of muscle metabolism, as PP1, establishing a connection with the well-known effect of red-IR laser radiation on the activity of cytochrome oxidase and ATP synthesis.4 Moreover, among the proteins overexpressed in the treated cells there were NLRP 10 and other proteins which regulate the inflammatory response and could contribute to the anti-inflammatory action attributed to laser therapy. Finally, the increase of proteins involved in cell adhesion/migration, angiogenesis and axonogenesis fits with the possibility to induce by laser treatment mechanisms related to tissue repair processes.

In conclusion, this study reports for the first time a proteomic analysis of IR laser treated myoblasts, thus contributing with original results to shed light on molecular and cellular
mechanisms underlying the effect of laser therapy in muscle repair and recovery.

Acknowledgements

This work was supported by Ente Cassa di Risparmio di Firenze and by Fondazione Cassa di Risparmio di Pistoia e Pescia. The authors thank ASA srl, which has provided the MLS laser for the entire duration of the study.

Effect of MSL laser on myoblast cell line C2C12.

L. Vignali, F. Cialdai and M. Monici.
ASAcampus, ASA Res. Div., Dept. Clinical Physiopathology, University of Florence, Florence, Italy.

ABSTRAcT

laser is widely used in many medical fields and its effects are reported by several
studies in literature. Very important are the applications in sports medicine,
physical medicine and rehabilitation, based on the analgesic, anti-inflammatory
and anti-oedema effects of laser therapy, as well as the stimulating action on tissue
repair processes. in our study, we analyzed the effects of an advanced laser system,
the Multiwave locked System (MlS), on myoblasts in order to evaluate the effectiveness of this laser in promoting recovery of damaged muscle tissue. The MlS device consists of two synchronized diodes emitting at 808 and 905 nm, respectively. c2c12 murine myoblasts cell line was used as experimental model since it is a widely accepted model in muscle cells behavior studies.

Viability and proliferation was assessed after a single treatment as well as after 4
consecutive treatment (1 treatment/day).
No significant changes were observed in viability, while proliferation decreased
after 4 treatments. Moreover, we found an increased expression of MyoD, a key
factor in myoblasts maturation. changes in cytoskeleton organization, in particular the networks of actin microfilaments and microtubules, were also observed.
Decresed proliferation rate, increased MyoD expression and cytoskeleton rearrangement are consistent with myoblast differentiation. finally the expression of molecules
involved in the regulation of extracellular matrix (ecM) turnover (collagen i, MMP-2, MMP-9) was analyzed. after 4 treatments, collagen i expression showed a 14% increase while MMP-2 and MMP-9 decreased of 33% and 18%, respectively.

These results suggest that MlS treatment could affect ecM turnover shifting the
balance toward the production rather than to the degradation. in conclusion, our findings demonstrate that MlS treatment induces in muscle cells a biological response that could
favour muscle cell differentiation and the recovery of diseased muscle tissue. a deeper knowledge of the mechanisms underlying the effects described above and a greater understanding of the changes in the biological response to variations in instrumental parameters setting can lead to concrete improvements in treatment protocols. 

INTRODUCTION

Lasers are widely used in biomedicine. Sport medicine, physiatrics and rehabilitation are among the most important fields of application. Here the analgesic, antiinflammatory, anti-oedema and stimulating effects of laser therapy are used to favour tissue repair and function recovery. According to the literature, many factors can contribute to the stimulating effect.
The moderate vasodilation increases the supply of nutrients and growth factors. for
example, it has been demonstrated that low-level laser (lll) irradiation ( Ga-al-as
laser) promotes expression of fibroblast growth factor (fGf) in rat gastrocnemius
muscle recovering from disuse muscle atrophy [1]. fGf promotes angiogenesis
and lead to fibroblasts activation [2,3] which determines an increase of collagen
synthesis, essential for tissue repair and regeneration [4-6]. Neoangiogenesis is
crucial for ensuring oxygen and nutritional substances to new tissues and has a very
important role in muscle recovery [7,3] effects that induce a local increase
of nutrients, promote angiogenesis and influence the development of
inflammation can strongly affect the healing process and functional recovery
of the injured tissues. 

Another factor widely recognized as fundamental to the stimulating effect is
the red/infrared (ir) laser-induced increase in aTP production in mitochondria [7-
9]. after treatment with He-Ne laser, an increase in membrane potential and
consequent aTP production have been observed in isolated mitochondria [10].
Moreover, many authors found that red/ir lasers may promote cell proliferation
[4,11-13].

All these effects are consistent with the hypothesis that the recovery of injured
tissues can be accelerated through the application of suitable laser therapy.
Studies on nerve fibers regeneration showed that reconnection process of nerve cells is accelerated after laser treatment, leading to the regeneration of insensitive areas [15-17]. other studies have demonstrated a faster recovery of wound healing and bone fractures, as well as a marked reduction in infarct size and myocardial infarct.

Many studies report on effects of laser radiation on muscle homeostasis and
repair mechanisms in this tissue. In a recent study, using mice as experimental
model, the anterior tibial muscle previously damaged by a cryolesion has
been exposed to lllT (Gaalas laser, 660 nm). although a significant reduction
in recovery time was not recorded, an increase of collagen iV was found in the
treated muscles.

Another study on mice demonstrated that He-Ne laser irradiation (632.8 nm),
associated with physical exercise, reduced skeletal muscle inflammation, improved
the activity of superoxide dismutase and diminished the activity of creatine kinase.
Some authors found an increase in proliferation of muscle satellite cells. These cells, usually quiescent, can be activated by factors released by cells of the injured muscle. The satellite cells have the function of creating new fibers and replacing the necrotic ones.
in the frame of studies aimed at understanding the mechanisms by which laser therapy can promote the repair and functional recovery of skeletal muscle, here we report the results obtained investigating the effect of ir laser radiation on myoblasts.
as for any other radiation source, the main parameters for characterizing laser
emission are: power, frequency and wavelength. These ones, together with
the features of the irradiated tissues or samples, strongly affect the way the
radiation propagates into the tissue/sample and the consequent effects.
in our experiments, we chose as the laser source a Multiwave locked System (MlS) because we hypothesized that this laser system could be particularly suitable
for the treatment of skeletal muscle. 

In fact the system is characterized by two synchronized emissions with wavelengths
808 and 904, respectively. The two emissions are absorbed by different
mitochondrial complexes, therefore the MlS treatment can affect cellular energy
metabolism by acting on multiple sites in the respiratory chain at the same time.
radiation with λ = 808nm is absorbed by the cytochrome oxidase (complex iV) which
is considered as a principal photoacceptor in mammalian cells [29,30]. it is know
that the activation of this mitochondrial enzyme after absorbing a radiation in red/
near infrared (ir) promotes the production of aTP. The radiation with λ= 905
nm interacts with the complexes i, ii, iii, iV of the respiratory chain and succinate
dehydrogenase.  

Considering the emission wavelengths and tissue type (muscular tissue) optical
properties, it is possible to estimate MlS radiation which is expected to propagate
within the tissue a penetration depth of about 10 mm in this kind of tissue; this
means that still about 13% of initial power reaches a 20 mm depth. Therefore it is
possible to affirm that MlS radiation can interact with deep-located muscle tissue.
Moreover, since our previous data (not yet published) demonstrated that MlS
radiation is absorbed by collagen and polysaccharide biogels, which are models
of extracellular matrix, we hypothesized that the MlS treatment could also affect
cell behaviour by modification of the extracellular microenvironment.

MATERIAL AND METHODS

Cell cultures

Murine myoblasts have been cultured in Dulbecco’s Modified eagle’s Medium
supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine and
10% fetal bovine serum (fBS). 

Cells were incubated at 37°c in humidified atmosphere containing 95% air and 5% co2 in order to maintain a pH value between 7.3 and 7.5. when confluence has been reached, cells have been washed twice with PBS, then treated with a 0,05% trypsin solution and plated on 55 cm2 plates. all the reagents have been purchased from Sigma (chemical
co St louis, Mo, USa).

MLS Treatment

The laser source was a Multiwave locked System (MlS) provided by ASA s.r.l. (arcugnano, Vicenza, italy). The instrument consists of two assembled laser diodes, with synchronized emissions at 808 and 905 nm, respectively.
The diode with λ = 808nm may emit in continuous mode, with a power P = 1.1w, or pulsed mode with an average power Pa = 0.55w and a maximum frequency of
2000Hz.

The diode λ = 905 nm is characterized by a pulsed emission with a maximum
frequency of 2000Hz and an average power Pa = 60mw.
Therefore, the MlS emission can occur in different modes, according to the
operator’s choice: continuous Mode (continuous Mode operation, cw): diode with λ = 808 nm, continuous emission and diode with λ = 905 nm, pulsed emission. Pulsed mode
(Pulsed Mode operation): diode with λ= 808nm, pulsed emission with pulses
repetition frequency f808 (Max value 2000Hz) and diode with λ = 905nm, pulsed emission with pulses repetition frequency f905 = f808.

When frequency changes, the emission features allow the average power of the
905nm diode emission to change, while the average power of the 808nm diode emission does not change. in fact, when the frequency changes the 808nm diode
emission duration changes in proportion, in this way the average power remains the
same. it is the temporal distribution of the released energy which changes. with the same emission time (and spot sizes), the whole energy (808nm + 905nm) changes
when the set frequency changes.

For our experiments, cells have been plated on slides Ø of 13mm (5000 cells per slide) previously sterilized and put in multiwell (plates of 24 wells) to carry out the treatment. Each plate has been put in a holder which allowed an easy scanning of the samples. each scanning lasted 20s.
The treatment was repeated once a day for 4 consecutive days in sterile conditions.
The treated samples have been compared with controls maintained in the same
conditions, except for the exposure to MlS laser device.
The following treatment parameters have been applied: 8 min exposure to 1500Hz emission frequency. To calculate the energy given to each sample during a single treatment (e) it has been considered the following relation:

e = Pt· ( tt/ n) (1)

where n is the number of samples (8 in our experiment), tt is the treatment time,
Pt is the average power, estimated on the slide surface (132 mm2 ), equal to the
sum of the two laser sources contribution (Pt ~ 200mw). entering the data in the
formula (1), we obtain e ~ 12.0 J.

Cell viability

Cell viability after exposure to MlS was determined by a Trypan Blue assay. The
dye is capable of selectively penetrate into dead cells. after treatment, cells
are washed and detached with trypsin/eDTa for a few minutes. Then cells are
centrifuged and resuspended in a solution of PBS and Trypan Blue (dilution factor: 2)
and counted, after 5 min of incubation, using Neubeuer emocytometer.
Immunofluorescence after treatment the cells were fixed in cold acetone for 5 minutes and then washed with PBS without ca and Mg. After blocking unspecific binding with
PBS containing 3% bovine serum albumin (BSa), cells were incubated overnight at 4°c with the specific antibodies: anti-α actin, anti-collagen i, anti-α tubulin and anti-vimentin antibodies (chemicon int, Temecula, ca), anti-Myo D antibody (Santa cruz Biotechnology, Heidelberg, Germany), anti-MMP-2 and anti-MMP-9 antibodies (abcam, cambridge, UK). The cells were then incubated with the fiTc (fluorescein isothiocyanate) conjugated
specific secondary antibodies (specifically: anti-mouse igG for tubulin and Myo D
antibodies, anti-rabbit igG for collagen i and MMP-2 antibodies, anti-mouse
igM for vimentin antibody and antigoat for MMP-9 antibody) (chemicon int, Temecula, ca). cells incubated with anti-α actin antibody did not need incubation with the secondary antibody since a mouse anti-actin alexa fluor® 488 conjugated was used. Negative controls were obtained by omitting the primary antibodies. Samples were evaluated by
an inverted epifluorescence microscope (eclipse Te2000-e, Nikon, italy) with oil
immersion objective (cSi S fluor 100x, N.a. = 1.3) at 100x magnification and
imaged by a Hires iV digital ccD camera (DTa, italy). fluorescence excitation has
been achieved by selecting the 365nm emission line of a mercury vapor lamp
(HBo 100w, osram). about 30 cells from different fields have been imaged
for each slide.

Image processing

The image processing has been performed by using a specific program written
in the labView language (National istruments). By first obtaining a binarized
image, in which pixels corresponding to cells and those corresponding to the
background have been given the value of 1 and 0 respectively, the program is
able to distinguish the cell signal from the background; as a second step, it calculates
the average cell intensity by applying the binarized images to the original grayscale ones. it is then possible to compare the average fluorescence intensity of a first images set (control samples) with the intensity of a second one (treated samples).

Data processing

The experiment has been made three times to confirm the results. for each slide 30
images have been acquired and selected in a random way. The fluorescence intensity
of each field (analyzed with previously described method) has been expressed as
the average pixel intensity corresponding to the visualized cells. intensities
corresponding to the 30 acquired fields have been further mediated to give a final
value, whose error has been calculated as Standard Deviation (SD). The statistical
significance has been determined using the T-Student’s test (chosing p<0.05).

RESULTS

The aim of this study was to evaluate the effects of MlS treatment on muscle cells and to identify mechanisms possibly involved in the stimulation of tissue repair.

For our experiments, we used a murine myoblasts cell line (c2c12) widely accepted
as a model in muscle cells behavior studies.
In particular, the research focused on cell viability and proliferation, organization of
cell cytoskeleton, expression of MyoD, an early marker of muscle differentiation, and
proteins involved in the extracellular matrix turnover (collagen i, MMP2, MMP9).

Viability and proliferation

In order to verify the effect of the exposure to MlS emission on cell viability and proliferation, Trypan blue assays were carried out 24 h after the first treatment and 24 h after the fourth treatment. As shown in fig.1, in both cases, no
significant differences were observed between treated samples and controls as
regards cell viability, which resulted higher than 97.5% in all the samples.
Cell proliferation did not change significantly after the first treatment, but showed a decrease of the 25% after four treatments (fig.2) 

Cytoskeleton

The cytoskeleton is an important structure for the cell since it allows both movement and shape modifications and

Fig.1. C2C12 cell viability assessed 24 h after MLS treatment and 24 h after the fourth MLS treatments. (control vs. MlS). Data were obtained by Trypan Blue assay.

Fig. 2. C2C12 cell proliferation assessed 24 h after MLS treatment and 24 h after the fourth MLS treatment. (control vs. MlS). Data were obtained by Trypan Blue assay

has an important role in intracellular transport and signalling. The cytoskeleton
is mainly composed of three elements: actin microfilaments, microtubules and
intermediate filaments made of tubulin and vimentin, respectively.
The distribution of actin, tubulin and vimentin in myoblasts exposed to MlS treatments was studied by immunofluorescence microscopy and image processing.
Actin is modified by mechanical stimulation, in particular by physical stimulation. it can
be used as a sensitivity marker of the cells when exposed to physical factors.
Moreover, it is considered an important marker for muscle cells differentiation.
as shown in fig.3 (a,b), after MlS treatments, actin expression decreased
by about 13% and cleary changed the organization of the microfilament network. The microfilaments appeared more concentrated in perinuclear area.

Fig. 3. Expression of cytoskeleton components assessed by immunofluorescence microscopy. Actin expression in control (a) and cells exposed to MlS treatment (b). Tubulin expression in control (c) and cells exposed to MlS treatment (d). Vimentin expression in control (e) and cells exposed to MlS treatment (f).

The treated samples showed also changes in the cell morphology, which resulted
elongated, when compared with control samples. from a quantitative point of
view, the expression of tubulin, which is the main constituent of microtubules,
did not change following laser treatment.
However, as observed in the case of actin, a different organization of the microtubule
network has been observed: in fact, in control cells microtubules were organized
radially while in treated cells appeared randomly distributed. See fig.3 (c,d).
we did not find any significant effect of the treatment on vimentin, the protein
which form the intermediate filaments [fig.3 (e,f)].

Extracellular matrix

The extracellular matrix (ecM) is the noncellular component of a tissue. It has many functions depending on the composition.
For example, it acts as support and anchorage for cells and is a reservoir of
growth factors. cells bind to ecM via membrane proteins called integrins.
Through these molecular “bridges”, ecM

Fig. 4. Expression of extracellular matrix components assessed by immunofluorescence microscopy. Collagen i expression in control (a) and cells exposed to MlS treatment (b). MMP-2 expression in control (c) and cells exposed to MlS treatment (d). MMP-9 expression in control (e) and cells exposed to MlS treatment (f).

deformations can transmit mechanical stresses to the cells and affect cytoskeleton
organization; in the same way cells can induce changes in ecM.
The ecM turnover is a key factor in the repair process of traumatized muscle.
The main ecM protein is collagen, which forms very dense fibres. Different types of
collagen are present in the various tissues. Collagen i is the most abundant in the
human body. it can be found in tendon, muscle, endomysial fibrils, the organic
part of the bone tissue [38,39] and in the scar tissue. after exposure to MlS,
myoblast cultures showed a moderate (14%) but significant increase (p< 0,025)
in collagen i expression. fig.4 (a,b)

The homeostasis of the ecM is also regulated by proteins belonging to metalloprotease family (MMP), which are involved in ecM degradation and repair during normal physiological processes. These proteins are also involved in pathological conditions like arthritis . in myoblast cultures treated with MlS we analyzed the expression of matrix metalproteinase-2 (MMP-2) and matrix metalproteinase-9 (MMP9), which degrade collagen iV, one of the most abundant types of collagen in skeletal muscle. In comparison with control samples we found a decrease of expression of 33% and 18% respectively
fig. 4 (c,d and e,f).

Differentiation markers as above described, the data of our experiment revealed a decrease in proliferation but no significant changes in viability. Since this means that the MlS treatment does not induce cell damage, we hypothesized that the reduction in the
growth rate could be due to the triggering of a differentiation process. Therefore, we analysed in the treated cells the expression of the differentiation marker MyoD. The
differentiation markers are molecules which are expressed when cells pass from
proliferation to maturation. each tissue has its own differentiation markers. MyoD,
an early marker of myogenesis, belongs to a protein family known as myogenic
regulatory factors (Mrfs). The main MyoD function is removing cells from cellular cycle and blocking proliferation. it is mainly expressed in muscle cells, where it has an important function in regulating muscle differentiation. Our results demonstrate that MlS treatment induced an increase of the 26% in MyoD expression (fig. 5).

Fig. 5. myoD expression assessed by immunofluorescence microscopy. control (a) and cells exposed to MlS treatments(b)

DISCUSSION

The analysis of the data obtained by our experiments shows that the exposure
to MlS treatment, even if repeated over time, did not produce significant changes in cells viability, which never fell below 97.5%. The proliferation decreased moderately, but significantly, after 4 treatments.

In literature there are many studies concerning the effect of laser radiation on cell viability. The results are often controversial and depends on laser type and experimental models used. However our results are in accordance with those reported by ferreira et al. in a study on the effect of red/ir lasers on c2c12 cells, the same as our experimental model.

Recent studies carried out on different cell types showed that proliferation increased
after exposure to wavelengths ≤ 780 nm, while it decreased by irradiation at 810 nm.
Since the unchanged cell viability demonstrated the absence of acute cell damage, the slower rate of growth induced us to hypothesize that MlS treatment could
promote muscle cell differentiation. This hypothesis was indeed confirmed by the
increase in MyoD that we found in treated myoblasts. As above explained, MyoD is
an early marker of myoblast differentiation and plays a key role in the maturation of
muscle cell.

The analysis of cytoskeleton organization, made through immunofluorescence microscopy, has shown that MlS treatment induced a considerable reshape both
in microtubules distribution and in the network of actin microfilaments.
These data are in agreement with results we obtained previously in chondrocytes and
fibroblasts exposed to ir laser treatment and also with the studies of ricci et
al, where changes in organization of actin filaments and stress fibers formation
in endothelial cells of rabbit aorta (reac) subjected to lllT are described.
It is well know that important changes of the cytoskeleton can be inducted by physical
stimulation and laser radiation is not an exception. These changes can determine
important effects on cells behavior, since microtubules have a primary function in
regulating distribution and positions of intracellular organelles and actin is involved
in cell shape determination, and regulates the adherence/migration processes [50].
Moreover, in muscle cells, actin has a very important and significant function.
Finally, the transition from proliferation to differentiation, such as that observed after
MlS treatments, involves changes in cell morphology and therefore in cytoskeleton
organization. 

Indeed, it has been demonstrated that substances like phospholipase D induce
myogenic differentiation through a remodeling of actin cytoskeleton.
MlS treated samples showed also changes in expression of molecules which have
important functions in reshaping the ecM. collagen i expression increased, in
agreement with what other authors have found recently in tissues exposed to Gaalas
laser (λ = 808nm).
On the contrary, the expression of MMP-2 and MMP-9, involved both in migration and
myoblasts differentiation, diminished.
The moderate increase in collagen and reduction in MMP-2 and MMP-9 could
affect myoblasts migration and ecM formation.

In conclusion, the results we obtained on cell viability and proliferation, structural
changes of the cytoskeleton, MyoD, collagen i, MMP-2 and MMP-9 expression
demonstrate that MlS treatment does not affect myoblast viability but can affect
migration, differentiation and production of ecM molecules.
These results indicate that MlS treatment is able to induce, in muscle cells, a
biological response that can affect muscle function. This response is consistent with
therapeutic effects observed at systemic level and suggest that MlS therapy could
be effective in treating muscle diseases by direct action on myoblast behaviour.
Additional studies to further understand the molecular mechanisms underlying
the observed effects are needed, since a better understanding of mechanisms and
biological responses evoked by use of different instrumental parameters can lead
to significant improvements in therapeutic protocols.  

Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

Monica Monici · Francesca Cialdai ·
Giovanni Romano · Paola Antonia Corsetto ·
Angela Maria Rizzo · Anna Caselli · Francesco Ranaldi

Received: 29 November 2011 / Accepted: 15 October 2012
© Springer Science+Business Media Dordrecht 2012

Abstract 

Microgravity-induced muscle atrophy is a problem of utmost importance for the impact it may have on the health and performance of astronauts.
Therefore, appropriate countermeasures are needed to prevent disuse atrophy and favour muscle recovery. Muscle atrophy is characterized by loss of muscle mass and strength, and a shift in substrate utilization from fat to glucose, that leads to a reduced metabolic efficiency and enhanced fatigability. Laser therapy is already used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of the research we present was to get insights on possible benefits deriving from the application of an advanced infrared laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of the hypotrophic tissue. The source used was a Multiwave Locked System (MLS) laser, which combines continuous and pulsed emissions at 808 nm and 905 nm, respectively. We studied the effect of MLS treatment on morphology and energy metabolism of C2C12 cells, a widely accepted myoblast model, previously exposed to microgravity conditions modelled by a Random Positioning Machine. The MLS laser treatment was able to restore basal levels of serine/threonine protein phosphatase activity and to counteract cytoskeletal alterations and increase in glycolytic enzymes activity that occurred following the exposure to modelled microgravity. In conclusion, the results provide interesting insights for the application of infrared laser in the treatment of muscle atrophy.

Keywords Muscle atrophy · Microgravity ·
Myoblasts · IR laser

Introduction

Aging and disuse, as occurs in bed rest and spaceflights, induce in skeletal muscle a reductive remodelling and may lead to atrophy. The mechanisms underlying muscle atrophy caused by disuse and muscle aging have some similarities: in both the lack of mechanical stimuli plays a relevant role. In this aspect, conditions associated with muscle disuse, such as the exposure to a weightless environment, are considered a model for
studying aging processes in skeletal muscle (Biolo et al. 2003), although other important factors such as changes in the innervation (Doherty 2003) and levels of cytokines and growth factors (Degens 2010) are involved in aging.
Disuse atrophy has been widely studied and is considered a problem of utmost importance in manned spaceflights. It is characterized by loss of muscle mass, force and power, changes in fiber type composition and increased muscle fatigue due to reduced metabolic efficiency: a shift in substrate utilization from fat to glucose occurs, leading to an enhanced fatigability (Fitts et al. 2000; Stein and Wade 2005; Blaauw et al. 2010).
Hexokinase (HK) activity, considered a marker of glycolytic metabolism, significantly increases (Manchester et al. 1990; Chi et al. 1992). The susceptibility of skeletal
muscle to damage increases and becomes particularly evident during postflight reloading (Fitts et al. 2000).

In the future, the increase of mission duration from one side and, from the other side, the expected increase of extravehicular activities, which could require sustained work output, will further exacerbate the problem of managing muscle atrophy during spaceflights and
postflight. Therefore, appropriate countermeasures are needed to prevent disuse atrophy and/or favour muscle recovery.
Several studies demonstrated the utility of physical protocols as vibration or electrical stimulation (Chopard et al. 2009; Guo et al. 2012), but too few data regarding the effects of different physical countermeasures on the processes involved in skeletal muscle
disuse atrophy and recovery are available.

Due to the proven ability of red-infrared (IR) radiation to enhance cell energy metabolism (Silveira et al. 2009) and reduce inflammation (Rizzi et al. 2006), laser therapy is already used in physical medicine, rehabilitation and sports medicine to accelerate muscle recovery (dos Santos et al. 2010) and to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise (Leal Junior et al. 2009).
This paper reports the results of a study aimed at investigating the effects of IR laser radiation on myoblasts and considering the possibility to apply IR laser therapy to promote muscle regeneration and recovery in disuse atrophy. We used as an experimental model the C2C12 skeletal muscle cell line, derived from satellite cells. The C2C12 cells are widely accepted as a model to study the behaviour of satellite cells (Burattini et al. 2004), which play a crucial role in skeletal muscle regeneration and repair (Wang and Rudnicki 2012) and are capable to repopulate atrophied muscle (Hawke
and Garry 2001). Moreover, C2C12 cells have been already used in previous studies regarding the effects of microgravity on mechanical signalling mechanisms in muscle plasticity (Torgan et al. 2000) and myoblast behaviour (Slentz et al. 2001; Pache et al. 2010).

Recently, studies in progress in our laboratory, aimed at understanding the cellular and molecular mechanisms underlying the effects of IR laser radiation on repair processes in muscle tissue, demonstrated that C2C12 cells exposed to IR laser radiation show enhanced cell energy metabolism and a significant increase in serine/threonine protein phos phatases (PSPs), in particular serine/threonine protein phosphatase 1 (PP1), which plays a crucial role in the regulation of glycogen metabolism and is involved in
myosin dephosphorylation, thereby controlling muscle contraction/relaxation (Cohen 2002; Ceulemans and Bollen 2004). Moreover, we observed that proteins
involved in cytoskeleton organization/cell shape regulation and muscle contraction, such as vimentin, actin and tropomyosin, also increased (Monici et al. 2012).
Following these findings, we hypothesized that IR laser radiation could be a useful tool to counteract the microgravity-induced impairment of energetic metabolism (Fitts et al. 2000), cytoarchitectural alterations (Pache et al. 2010) and decrease in the levels of contractile proteins (Torgan et al. 2000) observed in myoblasts exposed to microgravity.
Therefore, in myoblasts previously exposed to modelled microgravity, we tested the effect of IR laser treatment on cell metabolism and morphology.

Materials and Methods

Cell Culture

Murine myoblasts (C2C12 cell line) were routinely cultured in growing medium consisting of Dulbecco’s Modified Eagle’s Medium supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine and 10 % fetal bovine serum (FBS). Cells were incubated at 37 ◦C and 5 % CO2. All the reagents have been purchased from Sigma Chemical Co. (St
Louis, MO, USA).

MLS Laser Treatment

The treatments have been performed with an advanced Multiwave Locked System (MLS) laser (ASA Srl), that is a high power (average power up to 1.1 W, class IV) IR laser with two synchronized sources (laser diodes). The two modules have different wavelengths, peak power and emission mode. The first one is a pulsed diode laser, emitting at 905 nm, with peak optical power = 25 W; each pulse is composed of a pulse train (single pulse width = 100 ns, maximum frequency 90 kHz), thus varying the average power delivered to the tissue.

Frequency of the pulse trains may be varied in the range 1–2000 Hz. The second laser diode (808 nm) operates in continuous mode (P 1.1 W) or in pulsed mode (pulses repetition rate 1–2000 Hz), mean optical power output = 550 mW, duty ratio 50 % independently of the pulse repetition rate. The two propagation axes are
coincident.

For the treatment, cells were seeded in the central 8 wells of a 24-multiwell plate. The plate was placed inside a plexiglass support, specifically designed and built.
On the top of the support there was a central groove in which laser handpiece slided. The plate was perfectly aligned with the handpiece, at a distance of 3 cm from it, so that the spot formed by the two superimposed laser beams had a diameter equal to that of a single well (13 mm). The support allowed us to perform an homogeneous scan of 8 samples at the same time, by moving the spot at a constant horizontal velocity above the 8 treated wells (5.6 cm/s: each scan of 8 wells lasted 20 s), in order to have the same radiant energy impinging into each well (∼68 J for the whole treatment). Treatment
parameters were: 1500 Hz frequency, 8 min total scan time. The scan mode is now extensively used also in clinics because it allows to treat large areas and further
contributes to avoid photothermal damage. The treatment was repeated once a day, for 3 consecutive days in sterile conditions. The treated samples were compared with controls maintained in the same conditions, except for the laser exposure.

Random Positioning Machine 

A Random Positioning Machine (RPM) (Dutch Space, Leiden, The Netherlands) was used in order to model unloading conditions.
In the RPM, introduced by Hoson et al. (1997), samples are fixed close to the centre of two frames rotating one inside the other, driven by separate motors. The rotation of each frame is random and autonomous under computer control. The low g conditions are modelled by averaging the gravity vector via the independent rotation of the two frames.
In our experiments, the speed of rotation was 60◦/s (about 10−3 × g), and direction and interval were set at random. Temperature was maintained at 37 ◦C. The cells were placed in suitable T25 flasks, which were completely filled with culture medium in order to avoid
shear stress, and exposed to the RPM for 72 h.

Study Design

The following samples were prepared, analyzed and compared:

• Samples exposed to the RPM were compared with the corresponding ones non exposed to the RPM (1 × g controls). 
• Samples exposed to the RPM and then treated with MLS laser were compared with samples exposed to the RPM but untreated with MLS laser.
• 1 × g controls untreated and treated with MLS laser.

1 × g controls were placed on the fixed base of the RPM, facing the same vibrations and temperature as the rotating ones. At the end of the treatments, the cells were recovered and prepared for analytical tests.

Immunofluorescence Analysis

At the end of the experiments, cells were fixed for 5 min in cold acetone, then washed in phosphate buffered saline (PBS). After blocking unspecific binding with
PBS containing 3 % bovine serum albumin, cells were incubated overnight at 4 ◦C with the specific antibodies: anti-α actin, anti-tubulin and anti-vimentin. The cells were then incubated with the fluorescein isothiocyanate (FITC) conjugated specific secondary antibodies (specifically: anti-mouse IgG for anti-tubulin antibody and anti-mouse IgM for anti-vimentin antibody). Cells incubated with anti-α actin antibody did not need incubation with the secondary antibody since a mouse anti-actin Alexa Fluor® 488 conjugated was used. All antibodies were purchased from Chemicon Int, (Temecula, CA). Negative controls were obtained by omitting the primary antibodies. Samples were evaluated by an epifluorescence microscope (Nikon, Florence, Italy) at 100× magnification and imaged by a HiRes IV digital CCD camera (DTA, Pisa, Italy).

Cell Lysis

Cells were quickly rinsed in ice-cold phosphatebuffered saline (PBS, 10 mM sodium phosphate and 0.15 M NaCl, pH 7.2), and frozen at −80 ◦C. After thawing the material at room temperature, the lysis was performed at 4 ◦C in 50 mM Tris, pH 7.4, containing
5 mM ditiothreitol and Sigma protease inhibitors mix (1/100, v/v). After 30 min of incubation on ice, lysates were sonicated (three short bursts) and centrifuged
at 12,000 g in a microcentrifuge at 4 ◦C for 30 min.

Supernatants were quantified with respect to protein content by Bradford method.
Determination of Total Protein Concentration Total protein concentration was determined according Reagents) produced by Sigma Chemical Co. (St Louis,
MO, USA).

Determination of Pyruvate Kinase Activity

Pyruvate kinase (PK) activity was determined at 37 ◦C according to Hess and Wieker (1974), with slight modifications, continuously following NADPH oxidation at 340 nm, by using an UV-2100 spectrophotometer (Shimadzu, Columbia, MD). The assay mixture
contained in 1 ml final volume consisted of 50 mM triethanolamine (pH 7.6), 8 mM MgSO4, 5 mM EDTA, 75 mM KCl, 1.5 mM ADP, 0.15 mM NADH, 5 mg/ml
lactate dehydrogenase. 

The reaction was started by adding the substrate (0.8 mM phosphoenolpyruvate). The value of 6.22 mM−1 cm−1 is considered to be the NADH (or NADPH) molar extinction co-efficient. One unit of activity is defined as the quantity of enzyme which
transforms 1 μmole of substrate in 1 min, at 30 ◦C.

Determination of Hexokinase Activity

HK activity was determined at 37 ◦C according to Bergmeyer (1974), with slight modifications, continuously following the formation of NADPH at 340 nm, by using an UV-2100 spectrophotometer (Shimadzu, Columbia, MD). The assay mixture contained in 1 ml
final volume consisted of 50 mM triethanolamine (pH 7.6), 8 mM MgSO4, 5 mM EDTA, 1.5 mM ATP, 0.2 mM NADP, 2 mg/ml glucose-6-phosphate dehydrogenase. The reaction was started by adding the substrate (0.4 mM glucose).

Determination of Serine/Threonine Protein

Phosphatase Activity

Protein serine/threonine phosphatase (PSPs) activity was determined using p-nitrophenyl phosphate as a substrate. The substrate (4 mM) was dissolved in 25 mM Tris-HC1 buffer, pH 7.2, containing 5 mM ditiothreitol, 20 mM sodium-potassium DL-tartrate, and 0,1 mM Sodium orthovanadate. Tartrate and orthovanadate were added in order to inhibit protein tyrosine phosphatases, lysosomal acid phosphatases and non-specific phosphatases (Walton and Dixon 1993).

The reaction was stopped with 0.1 M KOH and the released p-nitrophenolate ion was measured by reading the absorbance at 400 nm (ε = 18,000 M−1 cm−l). The
activity measured in these conditions was completely inhibited by 10 μM cantharidic acid, a specific and strong inhibitor of all PSPs (Knapp et al. 1998).to the Bradford’s method (1976), using a kit (Bradford

Statistics

Experiments were carried out in triplicate. For immunofluorescence analysis, at least 30 cells per slide were scored in 10 random fields/slide, and the data were expressed as mean ± SD. Statistical significance was determined using a Student’s t test. A p value lower than 0.05 was considered statistically significant.

Results

In all the samples, the total protein content was analyzed and then used to normalize the measurements of enzymatic activities. The results show that the 1 × g control samples, both laser-treated and untreated, had a similar protein content. The protein content was reduced of about 40 % in the samples exposed to the RPM, but increased fivefold in samples exposed to the RPM and then treated with MLS laser (Fig. 1).

The HK activity, considered a marker of glycolytic metabolism (Grichko et al. 2000), did not show significant changes in 1 × g controls exposed to MLS laser, in comparison with untreated 1 × g controls. Myoblasts kept on the RPM showed a HK activity sevenfold
higher than 1 × g controls, but in the samples exposed to modelled microgravity and then to MLS laser irradiation the enzyme activity strongly decreased (Fig. 2a).
Also PK activity increased (30 %) in modelled microgravity and decreased (84 %) after the laser treatment (Fig. 2b). Serine/threonine protein phosphatase (PSPs)

Fig. 1 Analysis of total protein content—1 × g control samples, both laser-treated and untreated, showed a similar protein content. It decreased significantly in the samples exposed to the RPM for 72 h, but strongly increased in samples exposed to the RPM
and then treated with MLS laser. The symbol “*” indicates a p value lower than 0.05

Fig. 2 a Hexokinase activity—The HK activity did not change significantly in the 1 × g controls exposed to MLS laser, in comparison with the untreated ones. Myoblasts exposed to modelled microgravity (RPM) showed an impressive increase in HK activity. In the samples exposed to modelled microgravity, and then to MLS laser irradiation, the enzyme activity decreased to values

activity appeared more than doubled in laser-treated 1 × g controls, in comparison with the untreated ones.
In C2C12 cells exposed to gravitational unloading PSPs activity dramatically fell down, but went up significantly in cells treated with MLS laser after the exposure to
modelled microgravity conditions (Fig. 3).
The morphological analysis of the three major components of cytoskeleton, actin microfilaments, microtubules and intermediate filament network, which was performed by immuno-fluorescence microscopy, showed evident architectural alterations of all the three

Fig. 3 Serine/threonine protein phosphatase activity—PSPs activity increased in laser-treated 1 × g controls, in comparison with the untreated ones. In C2C12 cells exposed to gravitational unloading, PSPs activity dramatically decreased, but increased
significantly in cells treated with MLS laser after the exposure to modelled microgravity conditions. The symbol “*” indicates a p value lower than 0.05

lower than 1 × g controls. b Pyruvate Kinase activity—Also PK activity increased (30 %) in modelled microgravity and decrease (84 %) after the laser treatment. No differences were observed between the 1 × g controls untreated and treated with MLS laser.
The symbol “*” indicates a p value lower than 0.05

cytoskeletal structures examined in the samples kept in the RPM: in comparison with 1 × g controls, the actin expression in the cytoplasm decreased while stress fibers became more evident and microspikes with high actin expression appeared on the cell surface (Fig. 4c); the intermediate filaments lost the orderly perinuclear arrangement and concentrated at the center of the cell partially covering the nucleus (Fig. 4g), microtubules
lost the usual radial distribution starting from the organization centre but formed a tangled network (Fig. 4k).

No significant differences were found between lasertreated and untreated 1 × g controls (Fig. 4a, b, c, f,i, j). The samples exposed to modelled microgravity and then to the laser treatment showed a cytoskeletal structure restored and more similar to the 1 × g controls
than the samples kept in RPM without subsequent laser treatment (Fig. 4d, h, l).

Discussion

In literature, the data on protein content in muscle cells exposed to weightlessness are controversial and very difficult to compare because the authors used different
times of exposure (from minutes to days), different models (myoblasts, muscle fibers, 2D cultures or 3D cultures), real or modelled microgravity and, in this
last case, different devices for modelling microgravity (random positioning machine, rotating wall vessel).

Likely, the different results depend on the different experimental conditions and models used.
The decrease in total protein content we observed in the samples exposed to modelled microgravity conditions (Fig. 1) could be due to many processes: an 

Fig. 4 Cytoskeleton components analyzed by immunofluorescence microscopy—Samples exposed to modelled microgravity (c, g, k), compared to 1 × g controls, showed
evident architectural alterations of the three major cytoskeleton components: actin microfilaments, intermediate filaments and microtubules. Actin stress fibers became more evident and microspikes with high actin expression appeared on the cell
surface (c); the intermediate filaments lost the orderly perin uclear arrangement and concentrated at the center of the cell partially covering the nucleus (g), microtubules lost the usual radial distribution starting from the organization centre but formed a tangled network (k). No significant differences were found between untreated and laser-treated 1 × g controls (a, b, c,f, i, j). The samples exposed to modelled microgravity and then to the laser treatment showed a cytoskeletal structure restored and similar to the 1 × g controls (d, h, l)

increase in apoptosis, which often occurs in cell cultures exposed to weightless conditions (Uva et al. 2002;
Monici et al. 2006), a microgravity-induced decrease in myoblast proliferation, which has been recently found by other authors (Pache et al. 2010), altered protein synthesis/degradation (Moriggi et al. 2010), or a combination of these effects.
In agreement with data presented by other authors (Shimkus et al. 2011), preliminary experiments we performed did not reveal significant changes in proliferation and apoptosis induced by exposure to modelled microgravity. Some authors have found an increase (Slentz et al. 2001) or a decrease (Pache et al. 2010) in proliferation using different exposure times and conditions for simulating microgravity. Thus, in our experimental conditions, we are inclined to think that the decrease in total protein content depends on an altered protein synthesis/degradation.

As expected, a strong increase in HK activity was found in RPM-exposed cells and also PK activity increased (Fig. 2a and b). These results fit very well with data reported in literature: after spaceflights and bed rest, an upregulation of glycolytic enzymes, in
particular HK and PK levels, has been described by several authors (Chi et al. 1992; Stump et al. 1997; Stein et al. 2002) and represents a clear sign of microgravityinduced metabolic impairment. In myoblasts exposed to modelled microgravity, besides the decrease in total protein content and increase in glycolytic enzyme levels, the PSPs activity dramatically fell down (Fig. 3).
Despite the crucial role PSPs have in signal transduction and biological functions, the effect of the gravitational conditions on their expression and activity has been relatively little studied. PSPs, by opposing the action of protein kinases, regulate protein phosphorilation and therefore control metabolism and basical processes such as protein-protein interactions, gene transcription and translation, cell-cycle progression and
apoptosis, cytoskeleton dynamics and cell movement (Berridge 2009).

Our results suggest that the dysregulation of HK (and other glycolytic enzymes) could be related to the impressive reduction of PSPs activity, since it has been recently demonstrated in skeletal muscles of freezetolerant frogs that HK can be dephosphorilated by PP1 and, in this form, displays lower substrate affinity and
lower activity (Dieni and Storey 2011).

The deficit in PSPs activity could be also involved in the cytoskeletal alterations induced in myoblasts by weightlessness (Fig. 4c, g, k) and described also by other authors (Pache et al. 2010). In fact it is known that PSPs, and particularly PP1, are involved in actin
and actomiosin reorganization, regulation of cell shape and cell adhesion (Cohen 2002; Ceulemans and Bollen 2004).

Finally, a low PSPs activity could have altered protein turnover (Ceulemans and Bollen 2004), thus affecting protein content. MLS laser irradiation induced in 1 × g controls a
significant increase in PSPs activity (Fig. 3), confirming the results we obtained in preliminary studies on the effect of MLS emission on muscle cells, where we found
an significant increase in PSPs expression, foremost the expression of PP1 (Monici et al. 2012). In the same samples, protein content, HK and PK activities did not change significantly.
In myoblast cultures previously exposed to microgravity, the laser treatment was able to restore, at least partially, the level of PSPs activity (Fig. 3). Moreover, protein content strongly increased (Fig. 1) and both HK and PK activities returned to levels comparable to those expressed by the 1 × g controls, or even lower (Fig. 2a and b).
These results support the hypothesis that, in myoblasts exposed to modelled microgravity conditions, a relationship could exist between the downregulation of PSPs activity and alteration of metabolism markers. Moreover, as we hypothesized, in myoblasts previously kept in the RPM, MLS laser treatment, through the increase of PSPs activity, was able to reverse the metabolic alterations induced by modelled microgravity.
Our findings completely fit with the actual knowledge on the function of PSPs and, in particular, the role of PP1. In a recent review PP1 has been defined a “green” enzyme that promotes the rational use of energy and a reversal of the cell to a basal and/or
energy-conserving state, the recycling of protein factors and the return to basal patterns of protein synthesis;

in addition, PP1 plays a key role in the recovery from stress (Ceulemans and Bollen 2004).
The results obtained by analyzing cell morphology further confirm our hypothesis. In fact, as expected, C2C12 cells exposed to modelled microgravity showed an evident reorganization of cytoskeleton, with architectural features of the networks of microfilaments, microtubules and intermediate filaments very different from 1 × g controls. Conversely, myoblasts treated with MLS laser after RPM exposure exhibited a cytoskeletal architecture very similar to that of the 1 × g controls.
This faster return to the basic morphological pattern fits very well with the widely recognized role of PSPs in cytoskeletal rearrangement.
To the best of our knowledge this is the first time that a decrease in PSPs activity in myoblasts exposed to weightlessness is described and the ability of IR laser
radiation to reverse the effect is demonstrated.
In conclusion, the MLS laser treatment was able to restore the level of PSPs activity and to counteract increase in glycolytic enzymes activity and cytoskeletal alterations that occurred in C2C12 cells following the exposure to modelled microgravity.

While taking into account the limitation of the present data on myoblasts with respect to the interpretation for mature muscle fibers, however the results suggest that changes in PSPs activity could underlie some effects induced by microgravity in skeletal muscle
cells. Moreover, the possibility of reversing the effects by laser treatment opens the way to considerations about the usefulness of laser therapy to favour muscle recovery in disuse atrophy.
The findings of this “in vitro” study represent only a preliminary step in exploring the effectiveness of laser therapy as a countermeasure to disuse atrophy.
However, in our opinion, they provide original insights that encourage further research in this field.

Effect of NIR laser therapy by MLSMiS source against neuropathic pain in rats: in vivo and ex vivo analysi

Laura Micheli1, FrancescaCialdai2, Alessandra Pacini3, Jacopo JunioValerio Branca3,
Lucia Morbidelli 4, ValerioCiccone4, Elena Lucarini1, CarlaGhelardini1, Monica Monici2 &
Lorenzo Di Cesare Mannelli 1

Neuropathic pain is characterized by an uncertain etiology and by a poor response to common therapies. The ineffectiveness and the frequent side effects of the drugs used to counteract neuropathic pain call for the discovery of new therapeutic strategies. Laser therapy proved to be effective for reducing pain sensitivity thus improving the quality of life. However, its application parameters and efficacy in chronic pain must be further analyzed. We investigated the pain relieving and protective effect of Photobiomodulation Therapy in a rat model of compressive mononeuropathy induced by Chronic Constriction Injury of the sciatic nerve (CCI). Laser (MLS-MiS) applications started 7 days after
surgery and were performed ten times over a three week period showing a reduction in mechanical hypersensitivity and spontaneous pain that started from the first laser treatment until the end of the experiment. The ex vivo analysis highlighted the protective role of laser through the myelin sheath recovery in the sciatic nerve, inhibition of iNOS expression and enhancement of EAAT-2 levels in the spinal cord. In conclusion, this study supports laser treatment as a future therapeutic strategy in patients suffering from neuropathic pain induced by trauma.

Neuropathic pain is the result of damage (due to injury or disease) to the nervous system (including nerves), spinal cord and other central nervous system regions. Neuropathy patients suffer from spontaneous pain, allodynia (pain response to normally innocuous stimuli) and hyperalgesia (aggravated pain evoked by noxious stimuli) that interferes with their quality of life. Several experimental models have been developed to better understand neuropathy. The chronic constriction injury (CCI) model, developed by Bennett and Xie7, is a widely used model of mononeuropathy that replicates in rats most of the symptoms occurring in patients7–10.

Currently the most common way to treat pain is the administration of pain relief medications, although they have proved to be effective in only 30% of neuropathy patients which makes the research for new and effective treatments an ongoing challenge11–13.
The history of investigation and clinical use of laser therapy in medicine goes back to the late 1960s14. Since then, laser irradiation has been acknowledged as one of the most important non-pharmacological therapies.
Nowadays laser therapy use has become increasingly widespread because it is a non-invasive approach with few contraindications, rare side effects and relatively low costs, thus well accepted by patients15,16. The literature on laser therapy action mechanisms is extremely wide although with controversial findings that are difficult to compare and interpret, due to the very different experimental conditions (in particular the type of laser source and treatment parameters, that are wavelength, power, fluence, exposure time, etc…) used in the studies. However, through the years laser therapy has been has demonstrated its effectiveness in treating a number of different 1Department of Neuroscience, Psychology, Drug Research and Child Health - NEUROFARBA - Pharmacology and Toxicology Section, University of Florence, Florence, Italy. 2ASAcampus Joint Laboratory, ASA Res. Div. –
Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, italy. 

Figure 1. Laser treatment protocol with time schedule and parameters used. 

Figure 2. Monolateral neuropathy model induced by CCI. Sciatic nerve ligation was performed 7 days before the beginning of the test (day −7). Laser treatment [28 s, 30Hz; 50% int (mean power 1840 mW); peak power905 1 kW±20%; 5,147 J/cm2
; 51,4 J] was applied on days 1; 3; 6; 8; 10; 11; 13; 15; 17; 20; 22 at 0min and 30min after
laser application. The response to a noxious mechanical stimulus was measured by Paw pressure test. Values reported in the graph are referred to measurements conducted before treatments. Each value represents the mean±S.E.M of 6 rats per group performed in two different experimental sets. **P<0.01 vs sham group; °°P<0.01 vs CCI group pathological conditions17–24. 

The possibility to apply laser therapy in so many different pathological states depends on the effects that radiation has on important biological processes. A number of studies in literature have reported that laser radiation is effective in improving cell energy metabolism through ATP synthesis increase25–27. Further insights into the action mechanisms underlying enhanced cell energy metabolism were provided in a proteomic study, carried out on myoblasts exposed to near infraredb (NIR) laser radiation (808 and 905nm), where an increase in ATP-binding proteins and Protein Phosphatase 1
(PP1) was observed28. The same study, showed that laser irradiation induced a significant increase in NLRP10, an anti-inflammatory protein that inhibits the production of interleukin 1β28. The analgesic effect is also due to other mechanisms acting on the production of anti-nociceptive substances (endorphins), peripheral nerve conduction
and the transmission of nociceptive stimuli, as demonstrated by the rapid analgesic effect evoked by laser radiation in animal models of persistent pain18,29–31.
Preliminary studies showed that the anti-hypersensitivity effect and its persistence depended on treatment protocols and parameters (irradiation mode, treatment frequency, source wavelength and power, pulse frequency)31.

The aim of the present study was to investigate the effectiveness of a high power, dual wavelength NIR laser source (Multiwavelength Locked System laser, MLS-MiS) in producing a persistent anti-hypersensitivity effect in CCI-induced neuropathy caused by compressive damage in the rat. The laser therapy action mechanism were
also assessed with ex vivo evaluations of the central and the peripheral nervous system aimed to highlight the regeneration of the sciatic nerve and the reduction of the inflammatory processes in the spinal cord.

Results

Effect of laser treatments on CCI-induced hypersensitivity. Behavioural measurements were performed to evaluate the anti-hypersensitivity effect of repeated laser treatments on CCI-induced peripheral mononeuropathy in the rat. Laser treatment started one week after surgery and consisted of 10 sessions every other day, until the 3rd week (Fig. 1). The evaluation of hypersensitivity (Paw pressure test) was performed immediately
before and 30min after each laser application. Figure 2 shows the mean values monitored in the 3 groups of animals (sham, CCI, CCI+laser) before each of the 10 laser sessions.
The measurement performed before the first laser application (day 1) demonstrated that sciatic nerve ligation decrease the response to a mechanical noxious stimulus (Paw pressure test) from a value of 63.3±1.9 g (sham

Table 1. Response to a mechanical noxious stimulus of CCI+laser treated animals, Paw pressure test. Paw pressure test performed on CCI+laser treated animals, 30min after the daily laser treatment. Each value represents the mean±s.e.m of 6 rats performed in two different experimental sets.

Figure 3. Monolateral neuropathy model induced by CCI. Sciatic nerve ligation was performed 7 days before the beginning of the test (day −7). Laser treatment [28 s, 30Hz; 50% int (mean power 1840 mW); peak power905 1 kW±20%; 5,147 J/cm2
; 51,4 J] was applied on days 1; 3; 6; 8; 10; 11; 13; 15; 17; 20; 22 at 0min and 30min
after laser application. The hind limb weight bearing alteration was measured by Incapacitance test. Values reported in the graph are referred to measurements conducted before treatments. Each value represents the mean±S.E.M of 6 rats per group performed in two different experimental sets. **P<0.01 vs sham group; °°P<0.01 vs CCI group.

animals group) to 40.8 ±0.7 g (CCI and CCI+laser groups). In the CCI group this condition lingered for 3 weeks, until the end of the experiment (day 22). On the contrary, in the CCI+laser group two laser applications were enough to significantly increase the weight tolerated on the ipsilateral paw compared to the untreated CCI animals (48.8±1.3 g and 38.8±1.4 g, respectively, on day 6). The higher anti-hypersensitivity effect was recorded
after four laser applications (day 10), with a value of 60.0±2.5 g on the ipsilateral paw. Subsequent laser applications did not increase the paw threshold that remained stable (about 55 g) until the end of the experiment (day 22). In each laser session, the measurement performed 30min after laser irradiation did not show any significant
change in comparison to the pre-irradiation measurement (Table 1).

Monolateral pain as CCI induced alteration of hind limb weight bearing has been shown by the Incapacitance test (Fig. 3). Also in this case, measurements were performed immediately before each laser application and 30min later. The values monitored before each laser session are reported in Fig. 3. Before the first laser treatment
(day 1, 7 days after surgery), the difference between the weight burdened on the contralateral and the ipsilateral paw (Δ g) was significantly increased in both CCI and CCI+laser groups (about 60 g) compared to the sham group (−0.8±1.7g). In the CCI group this difference remained constant until the end of the experiment (day 22),
while in the CCI+laser group the gap has been reduced by about 50% on the first laser application (31.3±5.5 g, day 3). The pain relieving effect remained stable throughout the following laser treatments (days 6–16) to then slightly decrease from day 20. The measurements performed 30min after each laser session did not show significant changes compared to the pre-irradiation measurements (Table 2).

Table 2. Hind limb weight bearing alterations of CCI+laser treated animals, Incapacitance test. Incapacitance test performed on CCI+laser treated animals, 30min after the daily laser treatment. Each value represents the mean±s.e.m of 6 rats performed in two different experimental sets.

Effect of laser treatments on CCI-induced sciatic nerve damage: histological evaluation. The morphometric assessment by Luxol Fast Blue (LFB) tissue staining allowed to characterize the laser-dependent effect in comparison to the CCI-dependent alteration in the myelin sheath thickness. The histological examination of the specimens revealed a normal sciatic nerve appearance in the sham group with a regular distribution of
small and large diameter nerve fibers as well as a normal proportion between myelin sheath thickness and fiber diameter (Fig. 4, sham). As expected, the CCI group presented a wide distribution of very thinly myelinated nerve fibers (Fig. 4, CCI – black arrows), Wallerian degeneration (Fig. 4, CCI – black arrowhead) and unmyelinated
fibers in the tissue sections of the sciatic nerve 900 μm proximal to the ligation site, compared to sham rats. In contrast, nerves of animals treated with laser radiation (CCI+laser treated group) showed a remarkable myelin regeneration, as demonstrated by the presence of a greater number of nerve fibers that were surrounded by much
more myelin compared with the CCI group (Fig. 4, CCI+laser). Table 3 shows the measurements of fiber and axonal diameters and myelin thickness. Laser treatment significantly increased (°°P<0.01 vs CCI) the myelin thickness in comparison to CCI animals (2.24±0.18 vs 1.81±0.20) whereas fiber and axonal diameter measurements did not reveal any significant laser-dependent improvement.
Based on the results described above, to further investigate the effectiveness of NIR laser radiation in nerve protection and myelin sheath regeneration, Myelin Basic Protein (MBP) expression was evaluated by immunocytochemistry (Fig. 5). MBP is a major constituent of the myelin sheath produced by Schwann cells in the peripheral
nervous system. On day 22 (end of laser treatments, 30 days post-injury), MBP was significantly lower in the CCI group compared to the sham one (*P<0.01 vs sham). Laser treatment partially restored the MBP in the sciatic nerve of CCI+laser group (°P<0.01 vs CCI). These data further confirm the laser-dependent neuroprotection, in particular the restoration of the myelin sheet, revealed by morphometric analysis.

Effect of laser treatments on inflammatory markers. On day 22 (end of laser applications, 30 days post-injury), the nervous tissue (spinal cord) was evaluated for the expression of the glutamic acid transporter EAAT-2 and the inflammatory markers iNOS, COX-2 and mPGES-1. Results showed that while EAAT-2 was scarcely expressed in the spinal cord of sham animals, the CCI group (laser-untreated) presented an increased
expression, that was even higher in laser treated animals (CCI+laser group) (Fig. 6A).
A panel of inflammatory markers was then evaluated as the inducible isoform of NOS (iNOS) and the prostanoid pathway key enzymes COX-2 and mPGES-1. While COX-2 and mPGES-1 were not detected under any experimental condition (data not shown), the CCI procedure induced an increase in iNOS expression, whose levels were significantly blunted by laser application, with value even below the sham group (Fig. 6B). 

Discussion

Our previous studies showed the effectiveness of NIR laser therapy in reducing CCI-induced pain in the rat32.
A remarkable analgesic effect, whose peaked at about 30min after laser treatment, was obtained with a protocol consisting in the irradiation of two points, the first one directly located on sciatic nerve ligation and the other one on lateral side of the calcaneus (paw joint). However, the analgesia decreased rapidly (about 2hours later). In further studies, a more persistent analgesic effect was achieved when the irradiation of the two fixed points was followed by a scan on the whole leg, but even in this case the effect vanished 24hours later31.

Based on these evidences, in the present study a new protocol was designed and tested to obtain a long-lasting anti-hypersensitivity and protective effect through anti-inflammatory action and repair mechanisms. Results showed that laser treatment, performed by scans of the entire leg with the synchronized emission of a NIR, dual
wavelength, high power source (MLS-MiS), was able to control pain and inhibit the progression of a persistent painful condition. The loose ligation of the sciatic nerve induces a damage characterized by painful sensations correlated with overt tissue alterations. As previously reported, CCI model elicits a pain syndrome characterized
by mechanical and thermal hyperalgesia that begins about 3 days after nerve injury and reaches a plateau from 7 up to 30 days33. Laser treatment over these 3 weeks (10 sessions, every other day) counteracted the development of mechanical hyperalgesia already after two applications. The maximum anti-hyperalgesic effectiveness

Figure 4. Luxol Fast Blue staining. Representative micrographs of sciatic nerve axons in a sham, CCI and CCI+laser groups showing a partial laser-dependent neuroprotection of myelin thickness. Original magnification 400X. Scale bar=20 μm

Figure 5. Myelin Basic Protein (MBP) expression. A, Protein expression of MBP was evaluated by immunohystochemistry in each experimental group. CCI group and CCI+laser group were compared to each other and with sham group. Original magnification 400X. Scale bar=20 μm. B, AEC intensity was calculated by the integrated density of pixels for MBP. Control condition was arbitrarily set as 100% and results
are expressed as mean±S.E.M of 6 rats per group. Results are representative of at least three independent immunohistochemistry evaluations. *P<0.05 vs sham group and °P<0.01 vs CCI group. 

Each experimental point was performed in triplicate. Pictures are representative of fifteen field captured for each experimental point. *P<0.01 vs sham group; °P<0.01 vs CCI group was reached with 5 sessions, then the effect remained more or less stable until the end of the experiment. It is noteworthy that the measurements performed 30min after laser irradiation did not show significant differences compared to the pre-treatment ones. While, over the first 5 sessions, an evident increase in the pain threshold was recorded between each before-irradiation measurement and the measurements performed before and after the previous laser session (48h before). This means that the protocol used in this study does not induce an immediate analgesic effect, but rather a biological response rising more slowly but lasting longer over time.

The treatment protocol applied in this study was also able to reduce postural unbalance, a feature of neuropathy progression measured by hind limb weight bearing alterations. This measurement, in particular, may assess the somatosensory component of mononeuropathy highlighting spontaneous non evoked pain34. Laser treated
animals showed a halving of postural unbalance measurement from the first treatment and this effect remained constant until the end of the laser sessions, as the Incapacitance test showed. Also in this case, the measurements performed 30min after each laser irradiation did not show significant differences compared to the pre-treatmen

Figure 6. Western blot analysis of inflammatory markers. Spinal cords were isolated at the end of the experiment and proteins were run on SDS-PAGE. Proteins transferred on nitrocellulose membranes were then labelled with primary antibodies against EAAT-2 (panel A) and iNOS (panel B). β-actin normalization was performed f each sample. The graphs represent the means±S.E.M of 6 rats per group. *P<0.05 and **P<0.01
vs sham group; °°P<0.01 vs CCI group.

Table 3. Morphometric analysis of sciatic nerves. Morphometric analysis of sciatic nerves 29 days postsurgery showing the measurement of fiber and axonal diameter and of myelin thickness. Five µm sections stained with LFB were photographed at 100X magnification. The myelin thickness in photomicrographs taken from randomly selected fields was counted (6 rats/group) and analyzed using Origin 9.0 statistical software.
The results showed a significant decrease in axonal and fiber diameter as well as in myelin thickness in CCI compared to sham group (**P<0.01). Laser-treated group showed a significant increase in myelin thickness 

ones, confirming the paw pressure test results. The ex vivo analysis also highlighted a protective role of laser treatment in the central and peripheral nervous system.
In agreement with previous results33,35,36, histology, immunohistochemistry and levels of inflammatory markers (evaluated by western blot) showed that CCI-induced morphometric alterations of the sciatic nerve that dramatically affect the proximal distal from the injury. Besides, CCI mediated nerve architecture derangement is accompanied by local inflammatory reaction response, which include oedema, infiltration of hematogenous immune cells and induction of various soluble factors like cytokines, chemokines and small signalling molecules as nitric oxide. These findings were further confirmed by the increased expression of iNOS detected in spinal cord samples. Laser treatment significantly prevented the reduction in myelin sheath thickness and
hindered myelin degeneration, as highlighted by LFB staining and MBP immunohistochemistry. This result is in agreement with data reported by other authors, showing that NIR laser therapy was able to promote nerve fiber
regeneration and improve the quality of myelin layers in a rabbit model of peripheral nerve injury37. Moreover, previous data obtained using sources with emission 808nm and 904nm, the same wavelengths used in the present study, demonstrated that these NIR radiations enhanced nerve repair after end-to-side neurorrhaphy of the
median nerve in a rat model38 and increased HNRNPK expression in cell culture28. HNRNPK is a member of the heterogeneous nuclear ribonucleoproteins (hnRNPs) subfamily known to be required for axonogenesis during development and several of its RNA targets are under strong post-transcriptional control during the regeneration
process39.

The anti-inflammatory effect elicited by laser application in the CCI model was clearly demonstrated by the significant reduction in iNOS expression in the spinal cord. Also this result is consistent with previous studies28 that highlighted the increase of NLRP10 protein, an inflammasome inhibitor induced by laser radiation wavelengths

In the central nervous system, the excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft and extrasynaptic sites via glutamate reuptake into glial cells and neurons, allowing to keep its levels low and to terminate the synaptic transmission. Conditions that increase the levels of EAAT-2 expression, may
avoid an excess of glutamate capable of triggering a series of biochemical cascades associated to excitotoxicity and neuronal damage40. The findings of this study show that repeated laser treatments were able to strongly increase EAAT-2 levels, corroborating the anti-inflammatory and beneficial effects of the therapy on nervous and glial cells.
In conclusion, the results of this study indicate that NIR laser therapy carried out with MLS-MiS laser source and suitable protocols is able to control pain and prevent alterations of the nervous system induced by nerve injury.

While our previous studies31, reported a fast but not lasting analgesic effect was obtained with point by point irradiation, the protocol used in this study activated a slower but longer lasting biological response that counteracted hyperalgesia through three different mechanisms: (1) anti-inflammarory effect via inhibition of iNOS
expression; (2) repair effect through preservation/restoration of myelin sheath; (3) protective effect on central nervous system via enhancement of EAAT-2 levels. The collected data present a preclinical evaluation for a future therapeutic application of laser in patients suffering from neuropathic pain induced by trauma. The protocols can be further studied in order to exploit both the rapid analgesic effect that can be obtained by trigger point irradiation and the more persistent protective effect, with direct action on the cause of pain.

Materials and Methods

Animals. In all the experiments described below, male Sprague–Dawley rats (Envigo, Varese, Italy) weighing approximately 200–250 g at the beginning of the experimental procedure were used. Animals were housed in a Laboratory Animal Facility (CeSAL, Centro Stabulazione Animali da Laboratorio, University of Florence) and used one week after their arrival. Four rats were housed per cage (size 26×41 cm2), fed with standard laboratory diet and tap water ad libitum, kept at 23±1 °C with a 12 h light/dark cycle, light at 7 a.m. 

All animal manipulations were carried out according to the Directive 2010/63/EU of the European Parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes. The ethical policy
of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85–23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Italian Ministry of
Health (No. 54/2014-B) and from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines41. All efforts were made to minimize animal suffering and to reduce the number of animals used.

CCI-induced peripheral mononeuropathy. Neuropathy was induced according to the procedure described by7. Briefly, rats were anaesthetized with 2% isoflurane. Under aseptic conditions, the right (ipsilateral) common sciatic nerve was exposed by blunt dissection at the level of the mid thigh. Proximal to the trifurcation,
the nerve was carefully freed of the adhering tissue from the surrounding connective tissue, and 4 chromic catgut ligatures (4-0, Ethicon, Norderstedt, Germany) were tied loosely around the nerve with about 1-mm spacing between ligatures. After haemostasis was confirmed, the incision was closed in layers. The animals were allowed to recover from surgery and then housed one per cage with free access to water and standard laboratory chow.

Another group of rats were subjected to sham surgery in which the sciatic nerve was only exposed but not ligated. Laser treatment started 7 days after surgery.
Laser treatment and study design. Treatment were performed with a Multiwave Locked System laser (MLS-MiS, ASA S.r.l., Vicenza, Italy), a class IV NIR laser with two synchronized sources (laser diodes): the first one is a pulsed laser diode emitting at 905 nm wavelength, with peak power from 140W±20% to 1 kW±20% and pulse frequency varying in the range 1–2000Hz; the second laser diode emitting at 808 nm wavelength can
operate in continuous (max power 6W±20%) or frequenced (repetition rate 1–2000Hz, 50% duty cycle) mode.

The two laser beams work simultaneously, synchronously and the propagation axes are coincident.
Seven days after the sciatic nerve ligation, animals were randomly distributed into three groups:

1. Sham (n=6), animals subjected to sham surgery in which the sciatic nerve was only exposed but not ligated.
2. CI (n=6), animals subjected to the ligation of sciatic nerve, untreated with laser;
3. CCI+laser (n=6), animals subjected to the ligation of sciatic nerve, treated with laser. The treatment was performed 3 times a week over a 3 week period (days 1; 3; 6; 8; 10; 11; 13; 15; 17; 20; 22) for a total of 10 applications (Fig. 1) and consisted in a limb scan with the handpiece constantly moved over the treatment
   area. Irradiation was performed for 28 s with the following parameters: 30Hz; 50% int (mean power 1840 mW); peak power905 1 kW±20%; 5,147 J/cm2; 51,4 J

Paw pressure test. The nociceptive threshold in the rat was determined with ananalgesimeter (Ugo Basile, Varese, Italy) according to the method described by42. Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical mechanical probe. Mechanical pressure
was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. These limits assured a more precise determination of mechanical withdrawal threshold in experiments aimed to determine the effect of treatments. An arbitrary cut-of value of 100 g was adopted. Laser treatment started 7 days after injury (performed on days 1; 3; 6; 8; 10; 11; 13; 15; 17; 20; 22) and paw pressure test was conducted on the same days immediately before and 30min after laser treatment. The data were collected by an observer who was blinded to the protocol.

Incapacitance test. Weight bearing changes were measured using an incapacitance apparatus (Linton Instrumentation, UK) detecting changes in postural equilibrium after a hind limb injury43. Rats were trained to stand on their hind paws in a box with an inclined plane (65° from horizontal). The box was placed above the incapacitance apparatus. This allowed us to independently measure the weight that the animal applied on each
hind limb. The value considered for each animal was the mean of 5 consecutive measurements. In the absence of hind limb injury, rats applied an equal weight on both hind limbs, indicating a postural equilibrium, whereas an unequal distribution of the weight on hind limbs indicated a monolateral decreased pain threshold. Data are expressed as the difference between the weight applied on the limb contralateral to the injury and the weight applied on the ipsilateral one44. This behavioural measurement was performed on days 1; 3; 6; 8; 10; 11; 13; 15;
17; 20; 22 immediately before and 30min after laser treatment. The data were collected by an observer who was blinded to the protocol.

Tissue explants. On day 22, after the behavioural measurements, animals were sacrificed and the ipsilateral sciatic nerves were explanted. As previously reported, the portion containing the ligature was eliminated and a distance of 900 μm proximal to the ligation was chosen as optimal for evaluating the effect of laser treatment9.

Contralateral nerves were also dissected, and equivalent portions were collected. After fixation in 4% buffered neutral formalin solution, the tissue block was embedded in paraffin, then cut in a microtome to 5 μm thickness and mounted on positively charged slides. The spinal cord of each animal was also collected and frozen in N2 for
western blot analysis.

Luxol fast blue staininig. To perform the Luxol Fast Blue (LFB) staining, sections were immersed overnight in 0.1% LFB solution at 56–60 °C. After washing, differentiation was initiated by immersion in 0.05% aqueous lithium carbonate for 15 s followed by multiple immersions in fresh 70% ethanol, until white matter could be distinguished and nuclei decolorized. After washing, sections were immersed in 0.8% periodic acid for 10min
and then rinsed in distilled water. Finally, sections were incubated with Schiff ’s reagent for 20min and rinsed in distilled water for 15 min45.

The sections were semiquantified by an arbitrary score starting from 1, mild infiltrate and oedema, up to 10, severe infiltrate and widespread oedema. The morphometric analysis was conducted as previously reported33.
Sections were analyzed under light microscopy (100×magnification). At least 6 randomly distributed 20X fields within the transversal section of sciatic nerve were captured for each section. Images were examined using an Olympus BX40 microscope (Olympus, Milan, Italy) and photographed using a digital camera Olympus DP50
(Olympus, Milan, Italy). 

Myelin basic protein (MBP) immunohistochemistry. Slides were deparaffinized with xylene and rehydrated in ascending ethanol. Heat-induced epitope retrieval was performed for 3min in sodium citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0). After extensive washing in TBS (PBS+0.025% Triton X-100),
endogenous peroxidase was hindered with 0.3% H2O2 in 0.3% methanol for 15min. Sections were then blocked by incubation with Ultra V block (Thermoscientific, Milan, Italy) for 10min. MBP was detected using a mouse anti-MBP antibody (Chemicon, Milan, Italy) diluted 1:100 at 4 °C overnight. After washing in TBS, sections
were treated with an anti-mouse HRP-conjugated secondary antibody (1:1000, Invitrogen, Milan, USA) for 1h at room temperature. Development with 3-amino-9-ethylcarbazole (AEC) chromogen (BioOptica, Milan, Italy) was performed for 10 min at room temperature following the manufacturer’s instructions. After coverslipping,
protein expression was determined by image analysis of the slides on a Zeiss Axioimager microscope (Carl Zeiss; Jena, Germany) at 40×magnification.

Western blot of inflammatory markers in spinal cords. Protein extraction from spinal cords started with disruption and homogenization using the TissueLyser II (#85300 Qiagen). Samples were lysed on ice with CelLytic™ MT Cell Lysis Reagent supplemented with 2mM Na3VO4 and 1x Protease inhibitor cocktail for mammalian cells (Sigma Aldrich). Tissue lysates were centrifuged at 16000×g for 20minutes at 4 °C and the supernatants were then collected. Protein concentration was determined using the BCA protein assay kit (#23227 ThermoFisher Scientific)46. Electrophoresis (50μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [anti-EAAT2 (ab41621; dilution 1:1000) and anti-iNOS (ab49999, dilution 1:1000) purchased from Abcam, (Cambridge, UK); anti-COX-2 (AM05213PU-N, dilution 1:1000) was from Origene (Rockville, MD, USA); anti-mPGES-1 (Item No. 160140, dilution 1:200) was from Cayman Chemical (Ann Arbor, MI, USA)] and then detected by enhanced chemiluminescence system (BioRad, Hercules, CA, USA) Results were normalized to those obtained by using an antibody against β-actin (purchased from Merck KGaA, Darmstadt, Germany) diluted 1:1000047.

Statistical analysis. Behavioural measurements were executed on 6 rats per group (Sham, CCI, CCI+laser) performed in two different experimental sets. Measurements were taken in duplicate at least 1 min apart, the responses of both left and right paws were measured. Morphometric and immunohistochemical analyses were performed on 6 rats per group, evaluating four to five different sections of sciatic nerve per animal. Comparisons were carried out using Mann-Whitney nonparametric tests48. In all cases, the investigator was blind to the experimental status of each animal. Slides from control and experimental groups were labeled with numbers so that
the person performing the image analysis was blinded as to the experimental group. In addition, all images were captured and analyzed by an investigator other than the one who performed measures to avoid possible bias.
Results were expressed as mean (S.E.M) with One-Way analysis of variance (ANOVA). A Bonferroni’s significant difference procedure was used as a post hoc comparison. Data were analyzed using the “Origin 9.0” software (OriginLab, Northampton, MA, USA). Differences were considered significant at a P<0.05.

Acknowledgements

The authors acknowledge ASA Srl for providing the laser sources used in this study. This research was funded by the Italian Ministry of Instruction, University and Research (MIUR) and by the University of Florence.

Author Contributions

The authors’ contributions are as follows: L. Di Cesare Mannelli, C. Ghelardini and M. Monici designed the research, L. Micheli performed the in vivo experiments and wrote the manuscript, A Pacini and J.J.V. Branca performed the ex vivo analysis, L. Morbidelli and V. Ciccone performed the western blots and F. Cialdai and E.
Lucarini performed the laser treatments. F. Cialdai also drew all the components of the Fig. 1 that represents the laser treatment protocol with time schedule and parameters used.

Additional Information

Competing Interests: The authors declare no competing interests.
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Effect of NIR Laser Therapy by MLS‐MiS Source on Fibroblast Activation by Inflammatory Cytokines in Relation to Wound Healing

Shirley Genah 1,†, Francesca Cialdai 2,†, Valerio Ciccone 1, Elettra Sereni 2, Lucia Morbidelli 1,‡  and Monica Monici 2,*,‡

1. Department of Life Sciences, University of Siena, I‐53100 Siena, Italy; shirley.genah@student.unisi.it (S.G.); ciccone3@student.unisi.it (V.C.); lucia.morbidelli@unisi.it (L.M.)
2. ASAcampus Joint Laboratory, ASA Research Division & Department of Experimental   
   and Clinical Biomedical Sciences “Mario Serio”, University of Florence, I‐50139 Florence, Italy;
   francesca.cialdai@unifi.it (F.C.); elettra.sereni@unifi.it (E.S.)
   * Correspondence: monica.monici@unifi.it; Tel.: +39‐0552758366   
   † These authors contributed equally to this work.
   ‡ These authors contributed equally to this work.

Abstract: The fine control of inflammation following injury avoids fibrotic scars or impaired wounds. Due to side effects by anti‐inflammatory drugs, the research is continuously active to define alternative therapies. Among them, physical countermeasures such as photobiomodulation therapy (PBMT) are considered effective and safe. To study the cellular and molecular events associated with the anti‐inflammatory activity of PBMT by a dual‐wavelength NIR laser source, human dermal fibroblasts were exposed to a mix of inflammatory cytokines (IL‐1β  and TNF‐α)
followed by laser treatment once a day for three days. Inducible inflammatory key enzymatic pathways, as iNOS and COX‐2/mPGES‐1/PGE2, were upregulated by the cytokine mix while PBMT reverted their levels and activities. The same behavior was observed with the proangiogenic factor vascular endothelial growth factor (VEGF), involved in neovascularization of granulation tissue.

From a molecular point of view, PBMT retained NF‐kB cytoplasmatic localization. According to a change in cell morphology, differences in expression and distribution of fundamental cytoskeletal proteins were observed following treatments. Tubulin, F‐actin, and  α‐SMA changed their organization upon cytokine stimulation, while PBMT reestablished the basal localization.
Cytoskeletal rearrangements occurring after inflammatory stimuli were correlated with
reorganization of membrane α5β1 and fibronectin network as well as with their upregulation, while PBMT induced significant downregulation. Similar changes were observed for collagen I and the gelatinolytic enzyme MMP‐1. In conclusion, the present study demonstrates that the proposed NIR laser therapy is effective in controlling fibroblast activation induced by IL‐1β  and TNF‐α, likely responsible for a deleterious effect of persistent inflammation.   

Keywords: wound healing; NIR laser radiation; inflammation; fibroblasts; photobiomodulation

1. Introduction

Any injury or infection triggers an inflammatory reaction via cytokines deriving
from platelet degranulation and pathogen‐associated molecular patterns. Moreover, in
both cases, damaged cells release reactive oxygen species and non‐specic factors which
contribute to activate the inflammatory response in cells of the innate immune system,
fibroblasts, and epithelial and endothelial cells [1]. The induction of the inflammatory
response triggers a cascade of events mediated by recruitment, proliferation, and
activation of several cell populations, primarily immune and stromal cells, as well as

further release of cytokines, vasoactive factors, and growth factors that all together
contribute to the repair process [2,3].
Therefore, the correct progression of any acutely occurring inflammatory reaction is
a key factorin the path leading to successful healing, which consists in repair/regeneration
of damaged tissues and function recovery. However, the occurrence of alterations in the
finely‐tuned regulation of inflammation can cause pathologic conditions ranging from
healing delay (e.g., chronic ulcers) to fibrosis. Moreover, conditions of tissue stress or
altered function can induce an adaptive response, known as parainflammation or low
grade chronic inflammation, which is an intermediate condition between basal
homeostasis and acute inflammation, and is associated with serious diseases, including
obesity, diabetes, atherosclerosis, asthma, and neurodegenerative diseases [4].
Inflammation is regulated by a plethora of cell populations, biochemical, and
physical factors, but it is widely recognized that the cross‐talk between macrophages and
fibroblasts, and their ability to assume different phenotypes play a crucial role in
determining not only the evolution of inflammation, but also the subsequent stages of the
healing process.   
During inflammation, macrophages shift from a pro‐inflammatory phenotype (the
so called M1), characterized by massive production of pro‐inflammatory molecules, to an
anti‐inflammatory phenotype (the so called M2), which secretes suppressors of cytokine
signaling, passing through intermediate phenotypes [3,5–7].
In response to pro‐inflammatory mediators, resident fibroblasts or circulating
fibrocytes become the protagonists of the stromal activation and transdifferentiate in
myofibroblasts, their activated counterpart. Many pro‐inflammatory mediators are
implicated in fibroblast activation, migration, proliferation, and transdifferentiation,
including the cytokines tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), interleukin‐
6 (IL‐6), and the growth factors platelet derived growth factor (PDGF) and fibroblast
growth factors (FGFs). Activated fibroblasts and other mesenchymal cells engage a
crosstalk, which also reinforces the local immune response due to the induction of
vasodilation throught production of nitric oxide (NO) and prostanoids, and stimulates
angiogenesis via vascular endothelial growth factor (VEGF) production [8,9].   
In a normal evolution of the process, the turning off of the inflammatory response,
mediated by the shift of the macrophage phenotype from M1 to M2, opens the way to the
remodeling phase, which is dominated by fibroblasts through the production of extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs) [10]. A well‐timed resolution of inflammation is crucial for successful restoration of tissue architecture and function, while persistence of macrophage‐fibroblast activation state, with excessive production of pro‐inflammatory agents by fibroblasts and further recruitment of immune cells, leads to altered repair processes, from chronic wounds to fibrosis and scarring [11,12].

In summary, activated macrophages induce the stimulation of fibroblasts via
production of transforming growth factor‐β (TGF‐β), TNF‐α, IL‐1, and other cytokines. In
turn, activated fibroblasts can modulate the recruitment and behavior of immune cells via
release of cytokines and vasoactive factors as NO and prostanoids. Activated fibroblasts,
or myofibroblasts, regulate tissue remodeling by combining their ability to synthesize
ECM proteins and that of assuming contractile properties [13,14]. Contractile activity of
myofibroblasts increases ECM stiffness. In turn, ECM stiffness is, together with TGF‐β1,
among the most important factors in inducing myofibroblast differentiation and
persistence. Therefore, inflammation dysregulation can generate a feed‐forward loop with detrimental effects [15]. Therefore, the control of inflammation and fibroblast activation is crucial to obtain satisfactory morpho‐functional recovery and avoid defective healing.

Whatever the cause of inflammation (wound, trauma, infection), at the tissue level it
is characterized by redness, heat, oedema, pain, and loss of function.
In current clinical practice, a series of steroidal and nonsteroidal anti‐inflammatory
drugs can be used to control inflammation and the associated oedema and pain [16].
However, side effects, or even opposite effects on wound healing and other conditions inducing inflammation, limit their use, especially considering long‐term therapy, raising
the need for alternative countermeasures [16]. Moreover, anti‐inflammatory strategies
focused on a specific target (e.g., TNF‐α) did not produce the desired results [17]. Several
physical therapies and devices aimed to favor the healing process through the control of
inflammation and fibroblast behavior have been proposed, such as ultrasound, laser
therapy, electrical stimulation, and vacuum‐assisted closure [16,18,19]. Studies aimed at
elucidating the effectiveness of these therapies in controlling inflammation and the
deriving fibroblast activation might strengthen their use.

Laser therapy, currently called photobiomodulation therapy (PBMT), is one of themost widely applied to manage many different diseases characterized by acute or chronic
inflammation. The benefits of PBMT in terms of anti‐inflammatory [20], anti‐pain [21–23],
and anti‐oedema [24] properties are widely documented in literature. Moreover, PBMT
enhances cell energy metabolism and promotes anabolic and repair processes [25].
Further, PBMT has been shown to stimulate angiogenesis and collagen remodeling.

PBMT, being safe, non‐invasive, and non‐time‐consuming (short‐duration
application) is also well accepted by patients.   
Overthe last years, new molecularinsights into the action mechanisms of PBMT have
been obtained. In particular, it has been demonstrated that in chronic inflammatory
conditions, such as those related to periodontal diseases and osteoarthritis, PBMT is
effective in reducing the expression of pro‐inflammatory genes (TNF‐α, IL‐1β, IL‐6, IL‐8)
through the downregulation of NF‐ĸB signaling pathway via cAMP increase [27].
Another PBMT effect, which can be relevant in the evolution and outcome of the
inflammatory response, is to induce a decrease in matrix metalloproteinases (MMPs)
expression, as it has been recently demonstrated in an in vitro model of osteoarthritis.
MMPs are an important family of proteinases, able to degrade extracellular matrix
components and covering a broad range of tasks in inflammation, acquired immunity,
defense from injury and repair. MMPs are always present in acute and chronic,
physiological and pathological inflammatory processes, and experimental evidence
suggests that they can protect against or contribute to pathological evolution of
inflammation.

Despite the abundant literature on the ability of PBMT to control inflammation,
promote healing mechanisms, and counteract scarring, the effects that laser emissions
commonly used in PBMT exert on fibroblasts activated by a strong and persistent
inflammatory stimulation have not been clearly defined. In fact, for the most part, studies
used in vitro models of fibroblasts in the basal state.
Therefore, the present study was aimed at investigating the effect of PBMT on
activated fibroblasts. An in vitro model of fibroblasts, activated by exposure to
inflammatory stimuli, was characterized for morphological features, canonical
inflammatory and vasoactive cascades (inducible NO synthase and cyclooxygenase
(COX)/prostaglandin synthase enzymes), and outcomes on angiogenesis and ECM
remodeling. Then, the effectiveness and underlying molecular mechanisms of a high
power, dual wavelength NIR laser source in reducing fibroblast inflammatory phenotype
was investigated.

2. Materials and Methods

2.1. Cell Cultures

Normal human dermal fibroblasts (NHDF) were purchased from Lonza (Verviers,
Belgium) and grown in Fibroblast Growth Basal Medium (FBS; Lonza, Basel, Switzerland)
containing 10% Fetal Bovine Serum (FBS; Hyclone, Euroclone, Milan, Italy), 2 mM
glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Merck KGaA,
Darmstadt, Germany). Cells were cultured at 37 °C with 5% CO2 in Petri dishes and were
split 1:3 twice a week until passage 10.   

2.2. In Vitro Model of Inflammation

Cells (1 × 104) were seeded in 24‐multiwell plates and allowed to adhere (when
immunofluorescence analysis were planned, cells were seeded on 13 mm diameter glass
coverslips placed inside the 24‐multiwell plates). After 24 h, complete culture medium
was replaced by fresh complete culture medium supplemented with a mix of IL‐1β (10
ng/mL; #201‐LB/CF R&D System, Minneapolis, MN, USA) and TNF‐α (10 ng/mL; #201‐
LB/CF and #410MT, R&D System, Minneapolis, MN, USA). Cells were maintained with
the cytokines mix for 48 h. Control samples were treated in the same way, omitting the
cytokines mix.   

2.3. Laser Treatment

At the end of the 48 h of stimulation with cytokines mix, the medium of all samples
was replaced by a fresh complete culture medium. Then, samples which had been
previously stimulated with the cytokines mix were divided into two groups: A “treated
group”, that received laser irradiation and an “untreated group” that was not laser
irradiated. Laser treatment was performed with a Multiwave Locked System laser (MLS‐
MiS, ASA S.r.l., Vicenza, Italy) widely used in clinics. It is a class IV, NIR laser with two
synchronized sources (laser diodes): The first one is a pulsed laser diode emitting at
905 nm wavelength, with peak power from 140 W ± 20% to 1 kW ± 20% and pulse
frequency varying in the range 1–2000 Hz; the second laser diode emits at an 808 nm
wavelength and can operate in continuous (max power 6 W ± 20%) or frequent (repetition
rate 1–2000 Hz, 50% duty cycle) mode. The two laser beams work simultaneously and
synchronously, and the propagation axes are coincident.   

For laser exposure, only 6 wells of 24‐well plates contained cells. The wells
surrounding those with cells were filled with black cardboard to avoid light diffusion and
reflection. The exposure was performed by placing the plate inside a holder, which allows
the positioning of the laser handpiece at a 1.5 cm distance from the bottom of the wells, so that the spot of the two laser beams, impinging perpendicular to the sample surface, had the same diameter as a well (13 mm). Cells were irradiated for 10 sec with the following parameters: 10 Hz repetition rate; 50% int (mean power 1840 mW); peak power
1 kW ± 20%, fluence 5.19 J/cm2. All treatments were performed under laminar flow hood
at room temperature. The samples belonging to the untreated group were prepared and
kept under the same conditions used for the exposed samples, except for laser irradiation.

2.4. Experiment Design

The following samples were prepared, analyzed, and compared:   

1. Samples stimulated with a mix of IL‐1β and TNF‐α for 48 h and then exposed to 3
   laser treatments, repeated once a day, for 3 consecutive days under sterile conditions (CYKs + LASER Group);
2. samples stimulated with a mix of IL‐1β and TNF‐α for 48 h and not exposed to laser
   treatments CYKs Group);
3. samples not stimulated with a mix of IL‐1β and TNF‐α for 48 h and not exposed to
   laser treatments (CTRL Group).

For immunofluorescence analysis, an additional experimental group was included,
namely cells exposed to laser treatment alone (LASER Group).   
Six hours after the third laser treatment, all samples were prepared for the
subsequent analysis described in the following paragraphs.

2.5. Immunofluorescence Analysis

Cells grown on glass coverslips and treated as previously described, were fixed for 5
min with ice cold acetone. Unspecific binding sites were blocked with PBS containing 3%
bovine serum albumin (BSA; Sigma‐Aldrich, St. Louis, MO, USA) for 1 h at room
temperature. Then, cells were incubated overnight at 4 °C with specific anti‐NF‐kB (1:50; #sc‐372, Santa Cruz, Dallas, TX, USA), anti‐cyclooxygenase‐2 (COX‐2) (1:100; #TA313292,
Origene, Rockville, MD, USA), anti‐VEGF (1:50; #sc‐57496, Santa Cruz), anti‐α actin (1:100;
#MAB1501X, Millipore, Billerica, MA, USA), anti α‐smooth muscle actin (α‐SMA) (1:100;
#CBL171, Chemicon® by Thermo Fisher Scientific, Waltham, MA, USA), anti‐tubulin
(1:100; #05‐829, Millipore, Billerica, MA, USA), anti‐collagen I (1:100; #MAB3391,
Millipore, Billerica, MA, USA), anti‐fibronectin (FN) (1:100; #MAB1926‐I, Millipore,
Billerica, MA, USA), anti‐MMP‐1 (1:100; #MAB13439, Millipore, Billerica, MA, USA), and
anti‐α5β1 integrin (1:100; #MAB1999, Millipore, Billerica, MA, USA) primary antibodies
properly diluted in PBS with 0.5% BSA. After washing three times with PBS‐0.5% BSA,
samples were then incubated for 1 h at 4 °C in the dark with: Alexa Fluor 555™ conjugated secondary antibodies [specifically: Anti‐mouse IgG (#A‐21422, Invitrogen™ by Thermo Fisher Scientific) for anti‐NF‐kB and anti‐VEGF antibodies and anti‐rabbit IgG (#A‐21428, Invitrogen™ by Thermo Fisher Scientific) for anti‐COX‐2 antibody] and fluorescein
isoth iocyanate (FITC) conjugated specific secondary antibody [specifically: Anti‐mouse
IgG (#AP124F, Millipore) for anti α‐SMA, anti‐tubulin, anti‐collagen I, anti‐fibronectin,
anti‐MMP‐1, anti‐α5β1 integrin primary antibodies]. All secondary antibodies were
diluted 1:200 in PBS with 0.5% BSA. Cells incubated with anti‐α actin antibody did not
need incubation with the secondary antibody since a mouse anti‐actin Alexa Fluor® 488
conjugated was used. Again, samples were washed three times and then mounted on
glass slides using Fluoromount™ aqueous mounting medium (Sigma‐Aldrich St. Louis,
MO, USA) [31]. In samples of incubated anti‐NF‐kB, anti‐COX‐2, and anti‐VEGF, before
mounting, nuclei were marked with DAPI (#D9542, Sigma‐Aldrich, St. Louis, MO, USA)
diluted 1:5000 in PBS with 0.5% BSA for 30 min at room temperature. The fluorescent
signal of samples stained with anti‐NF‐kB, anti‐COX‐2, and anti‐VEGF antibodies was
acquired using a Leica TCS SP5 laser scanning confocal microscope (Leica, Wetzlar,
Germany). All other samples were evaluated by an epifluorescence microscope (Nikon,
Florence, Italy) at 100x magnification and imaged by a HiRes IV digital CCD camera
(DTA, Pisa, Italy). Based on the CCD images, a relative immunofluorescence
quantification was carried out by image analysis routines (ImageJ 1.53 analysis software,
National Institutes of Health, Bethesda, MD, USA) for samples stained with anti‐collagen
I and anti‐α5β1 integrin antibodies. After appropriate thresholding to eliminate
background signal and creation of a proper image mask, a pixel intensity histogram was
acquired.

2.6. Western Blot

Cells derived from the different experimental conditions, were detached from 24
multi‐well plates, collected in 15 mL tubes, and lysed with CelLyticTM MT Cell Lysis
Reagent supplemented with 2 mM Na3VO4 and 1X Protease inhibitor cocktail for
mammalian cells (Sigma‐Aldrich). Cell lysates were centrifuged at 16000× g for 20 min at
4 °C, and the supernatants were then collected. Protein concentration was determined
using the Bradford protein assay (Sigma‐Aldrich). Electrophoresis with equal amounts of
proteins (50 μg) was carried out in NuPAGETM 4–12% Bis‐Tris precast Gels (Thermo Fisher
Scientific) as previously reported [32].   
Proteins were transferred onto nitrocellulose membranes, blocked for 1 h in a PBS–
0.05% Tween solution (Sigma‐Aldrich) supplemented with 5% (wt/vol) of Blotting‐Grade
Blocker (Bio‐Rad, Hercules, CA, USA). Membranes were then incubated overnight at 4 °C
with the primary antibodies properly diluted in PBS–0.05% Tween solution supplemented
with 1% (wt/vol) of Blotting‐Grade Blocker: anti‐inducible NO synthase (iNOS) (1:250;
#sc‐7271, Santa Cruz), anti‐COX‐2 (1:1000; #160106, Cayman Chemical, Ann Arbor, MI,
USA), and anti‐microsomal prostaglandin E synthase‐1 (mPGES‐1) (1:500; #160140,
Cayman Chemical). Immunoblots were washed three times with PBS–0.05% Tween
solution and then incubated for 1 h with the respective species‐specific secondary
antibody conjugated with horseradish peroxidase HRP (Promega, Madison, Wisconsin,
US) diluted 1:2500 in PBS–0.05% Tween solution. The membranes were finally incubated with SuperSignalTM West Pico PLUS chemiluminescent Substrate (Thermo Fisher
Scientific), and the immunoreaction was revealed by ImageQuant LAS 4000
chemiluminescence system (GE Healthcare, Chicago, IL, USA). Results were normalized
to those obtained by using an antibody against β‐Actin (#A5441, Sigma‐Aldrich) diluted
1:10,000 in PBS–0.05% Tween solution.   

Immunoblots were analyzed by densitometry using Image J software, and the results,
expressed as arbitrary density units (A.D.U.), were normalized to β‐Actin.   
2.7. Immunoassays for Prostaglandin E‐2 and VEGF Quantification
Conditioned media were collected at the end of the experiment, frozen, and stored at
−80 °C until use. Prostaglandin E‐2 (PGE‐2) and VEGF levels were measured using ELISA
kit: Prostaglandin E2 ELISA kit‐Monoclonal (Cayman Chemical, Ann Arbor, Michigan,
US) and VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively, following
the manufacturerʹs instructions. Dosing of each sample was performed in double, and
PGE‐2 and VEGF levels were expressed as (pg/mL).   

2.8. Statistics

Three different experiments were carried out in triplicate. Data are reported as means
±SD. Statistical significance was determined using two‐sided Student’s t test. A p value
lower than 0.05 was considered statistically significant. For immunofluorescence analysis,
at least 30 cells per slide were scored in 10 random fields/slide.

3. Results

3.1. Set up of an “In Vitro” Inflammatory Model in Fibroblasts Cultures

The human dermal fibroblasts NHDF have been treated with a mix of cytokines (IL‐
1β and TNF‐α, each at 10 ng/mL) for 24 h and 48 h, then the occurrence of inflammatory
features depending on the exposure time has been evaluated.   
Microsomal PGE synthase‐1 (mPGES‐1), the pivotal inducible enzyme of the
prostanoid inflammatory pathway, was evaluated by western blot after 24 h and 48 h of
stimulation. A consistent rise in mPGES‐1 was observed after both 24 h and 48 h, the
increase being more evident at a longer time of exposure (Figure 1, upper panel, 0.8 ± 0.2
and 4.8 ± 0.9 fold increase of mPGES‐1 expression in the presence of cytokines with respect to control, at 24 h and 48 h, respectively). The up‐regulation of the mPGES‐1 enzyme generated a significant increase in the final product, prostaglandin E2 (PGE‐2), releasedby fibroblasts in the conditioned medium, documenting an activation of the enzymatic cascade (Figure 1, lower panel). Based on these results, the stimulation time of 48 h was chosen for further experiments.

Figure 1. Development of an inflammation model on normal human dermal fibroblast (NHDF) cells. Fibroblasts were treated with IL‐1β (10 ng/mL) + TNF‐α (10 ng/mL) for 24 h and 48 h. Whole cell lysates were collected to assess mPGES‐1 expression by Western blot (upper panel). Samples A and B represent intra‐experimental duplicates. The measurement of PGE‐2 performed by immunoenzymatic assay is reported (lower panel). At both times, there is an upregulation of the prostanoid system. Data represent means +/− SD (n = 3) *** p < 0.001 CYKs group vs. CTRL group.

3.2. Effect of Laser Treatment on Inflammatory Phenotype in Fibroblasts

3.2.1. Expression of Inflammatory Markers

In order to evaluate whether laser treatment could affect the inflammatory model
described above, samples, after the stimulation with cytokine mix, were exposed to laser
radiation according the following experimental protocol: NHDFs were treated with
cytokine mix (IL‐1β and TNF‐α, each at 10 ng/mL) for 48 h; then, culture medium was
replaced by fresh culture medium, and samples were divided into the following groups:

1. CYKs + LASER Group—samples previously stimulated with the cytokine mix and then
   exposed to laser treatment (3 treatments, repeated once a day, for 3 consecutive days); 
2. CYKs Group—samples previously stimulated with the cytokine mix and not exposed to laser treatment; 
3. CTRL Group‐samples not stimulated with the cytokine mix and not
   exposed to laser treatment. Six hours after the third laser treatment (T = 126 h), all the samples were recovered and iNOS, COX‐2 and mPGES‐1 protein expression was
   evaluated by western blotting.

Following the exposure to inflammatory cytokines, a significant up‐regulation of the
inflammatory enzymes was observed (Figure 2). The group stimulated with cytokines and
then treated with lasers showed a strong decrease in inflammatory enzyme expression
compared to the group stimulated only with cytokines. For iNOS and COX‐2, the decrease
reached statistical significance (Figure 2). 

Figure 2. Effect of laser treatment on NDHF cells stimulated with pro‐inflammatory cytokines.
Fibroblasts were treated with IL‐1β and TNF‐α, each at 10 ng/mL for 48 h, then culture medium was replaced by fresh culture medium and samples divided into 3 groups: CYKs + Laser—samples stimulated with cytokine mix and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days); CYKs—samples stimulated with cytokine mix and not exposed to laser treatment; CTRL—samples not stimulated and not exposed to laser treatment.
Six hours after the third laser treatment, whole cell lysates of all samples were collected, and Western blot was performed to assess protein abundance of iNOS, COX‐2, and mPGES‐1 (upper panel). Immunoblots were analyzed by densitometry and the results, expressed as arbitrary density units (A.D.U.), were normalized to β‐Actin (lower panel). Data represent means +/− SD (n= 2) * p < 0.05 and *** p < 0.001 CYKs group vs. CTRL group, # p <0.05 CYKs + Laser group vs. CYKs group.

To validate data obtained by Western blot, the main product of prostanoid enzymatic
cascade, PGE‐2, was measured in NHDF conditioned media recovered from the samples
6 h after the third laser treatment (T = 126 h). The cytokine mix‐stimulated fibroblasts
showed a significant increase in PGE‐2 released in the medium in comparison with control samples (209 pg/mL in basal condition and 11000 pg/mL after 48 h of stimulation with the inflammatory mix). Although the resulting data were not significant, laser exposur reduced PGE‐2 levels with a clear trend towards damping of the prostanoid pathway (Figure 3, upper panel).   

Additionally, conditioned media were assessed for the release of the angiogenic
factor VEGF, involved in neovascularization and granulation tissue formation. While
inflammatory stimuli significantly increased VEGF levels, laser exposure strongly
reduced VEGF availability in the medium, being the levels well below the basal,
unstimulated condition (Figure 3, lower panel).

Figure 3. Modulation of PGE‐2 and vascular endothelial growth factor (VEGF) release in NHDF cells exposed to pro‐inflammatory cytokines and laser treatment. Fibroblasts were treated with IL‐1β and TNF‐α, each at 10 ng/mL for 48 h, then the culture medium was replaced by fresh culture medium and samples divided into 3 groups: CYKs + Laser—samples stimulated with cytokine mix and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days);
CYKs—samples stimulated with cytokine mix and not exposed to laser treatment; CTRL—samples not stimulated and not exposed to laser treatment. Six hours after the third laser treatment, all samples were recovered, and PGE‐2 (upper panel) and VEGF (lower panel) levels were evaluated in conditioned media using specific ELISA kits. Dosing of each condition was performed in double, and quantification is expressed as pg/mL. Data represent means +/− SD (n = 2) ** p < 0.01 and *** p < 0.001 CYKs group vs. CTRL group, ### p < 0.001 CYKs + Laser group vs. CYKs group.

Ultimately, the modulation of the inflammatory response at the cellular level was
evaluated through confocal microscopy. Inflammation is a protective response
characterized by a series of reactions, such as vasodilation and recruitment of immune
cells to the site of injury. NF‐κB is an inducible transcription factor, responsible for the
activation of genes involved in this process, including COX‐2 and VEGF [33]. The
localization of the nuclear transcription factor NF‐κB and the intensity of the fluorescent
signal given by the expression of its downstream genes COX‐2 and VEGF were analyzed
in the samples described above. In control samples, the transcription factor seemed to
remain outside the nucleus, since the fluorescent signal was mainly cytoplasmic (Figure
4a). NHDF stimulation with IL‐1β  and TNF‐α  induced a consistent increase in the
expression of NF‐κB, as evidenced by a higher fluorescence intensity. Furthermore, in
many cells, a change in the localization was observed, with accumulation of the signal at
the nuclear level (Figure 4b; white arrows). In samples stimulated with the cytokine mix
and then treated with laser, a clear decrease in intensity of the signal linked to the
transcription factor was observed, although some cells with NF‐κB located in the nucleus (Figure 4c; white arrows) were still present. In cells treated with laser alone, the presence
of some NF‐κB punctuation at nuclear level was observed (Figure 4d)

Figure 4. Laser treatment reduces inflammatory response in NHDF cells by limiting NF‐κB translocation into the nucleus and down‐regulating COX‐2 and VEGF expression. Confocal analysis of NF‐κB (panels (a–d)), COX‐2 (panels (e–h)) an VEGF (panels (i–l)) expression and localization (magnification 63×) evaluated by immunofluorescence on NHDF in basal
conditions (CTRL; panels (a,e,i)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panels (b,f,j)), stimulated with IL‐1β and TNF‐α for 48 h, and then exposed to lasertreatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + LASER; panels (c,g,k)) and exposed to laser alone (LASER; panels (d,h,l)). For each series, the left panels show the protein of interest in red, while DAPI staining (blue) was merged on the right panels. White arrows indicate cells with nuclear localization of NF‐kB. Bar = 25 μm.

A similar trend was described for COX‐2. The enzyme expression resulted strongly
enhanced by the cytokine mix (Figure 4f) in comparison with unstimulated and laser alone controls (Figure 4e,h), where the fluorescence signal was weak and located in the
cytoplasm. In samples stimulated with the cytokine mix and then treated with laser, the
signal was similar to that observed in control (Figure 4g). However, in the last condition,
mixed cell populations were noticed, some still over‐expressing the enzyme and others
returned to control levels. Similarly, the VEGF signal also followed a modulation
superimposable to that of COX‐2, presumably dictated by the transcription factor NF‐kB.
VEGF labelling, not affected by laser alone (Figure 4k), increased by stimulation with
cytokines mix (Figure 4j). Following laser treatment, the cytokine‐induced VEGF intensity
completely returned to the basal levels (Figure 4l), confirming the data obtained by
protein dosage carried out on conditioned media, as previously illustrated (Figure 3,
lower panel).

3.2.2. Morphology and Cytoskeleton Organization

The stimulation with the mix of IL‐1β and TNF‐α produced a marked change in cell
morphology. The organization of the microtubules, which control cell architecture,
changed as well. In the control samples, fibroblasts were generally star‐shaped and spread on the substrate. The specific labeling for tubulin showed the well‐known radial
distribution of microtubules which branch off from a nucleation center(Figure 5a), usually
anchored at the centrosome and the Golgi apparatus [34]. In the stimulated samples, the
cells appeared spindle‐shaped, elongated, with a dense, longitudinal microtubule
network [34], where it was difficult to distinguish a nucleation center (Figure 5b). In the
samples first stimulated and then treated with laser, fibroblasts regained a shape similar to the controls, with a clearly distinguishable microtubule nucleation center and radially
organized microtubules (Figure 5c).

Figure 5. Effect of laser treatment on tubulin, α‐actin, α‐SMA expression, and distribution.
Microscopy analysis of tubulin (a‐c), α‐actin (d–f), and α‐SMA (g–i) expression evaluated by
immunofluorescence (magnification 100×) on NHDF in basal conditions (CTRL; panels (a,d,g)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panels (b,e,h)), stimulated with IL‐1β and TNF‐ α for 48 h, and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + Laser; panels (c,f,i)). Bar = 10 μm.

As regards actin distribution and organization, control fibroblasts showed a
perinuclear area rich of G‐actin, a network of very thin microfilaments distributed in the
cell cytoplasm, and a thin actin layer placed close to the plasma membrane (Figure 5d). In
the stimulated fibroblasts, F‐actin was predominant, with microfilaments arranged in
parallel and thicker in comparison with those observed in control cells (Figure 5e). As
already noted for tubulin, also in the case of actin, the stimulated cell samples which were
then exposed to laser radiation recovered a condition similar to the control cells, with G‐
actin thickened in the perinuclear area, few very thin microfilaments and a thin actin layer
close to the cell membrane (Figure 5f). 

Alpha‐smooth muscle actin (α‐SMA) is the actin isoform that predominates within
smooth‐muscle cells. Its expression generally increases in the transition fibroblast‐
myofibroblast. In fact, myofibroblasts acquire a contractile phenotype, which is
responsible for merging the wound edges in the healing process. Therefore, α‐SMA is
considered a marker of myofibroblast differentiation. In control samples, α‐SMA staining
revealed some stress fibers, which completely disappeared in fibroblasts stimulated with
IL‐1β  and TNF‐α, where the fluorescence signal coincided with the nucleus and was detectable only in the nuclear area (Figure 5g,h). The samples exposed to laser radiation
after the cytokine mix stimulation showed an intermediate situation. The signal in the
nuclear area was still detectable, but fibers organized in parallel appeared (Figure 5i).

3.2.3. Extracellular Matrix Proteins and Membrane Integrin

Integrins are cell surface receptors which control various cellular functions. Integrin
receptors connect the cell cytoskeleton with the ECM proteins, thus being involved in
signaling changes of the extracellular microenvironment and leading to cellular
responses. In particular, α5β1 integrin is a fibronectin receptor and has a well‐defined role in cell adhesion, migration, and matrix formation, which are functions of crucial
importance in physiological and pathological processes such as wound healing and
fibrosis. In the control samples, α5β1 clusters were located at focal adhesion points mostly in the perinuclear area, along cellular protrusions, and at their ends, generally arranged parallel to the major axis of the cells (Figure 6a). 

In the samples stimulated with the cytokine mix, the expression of the integrin significantly increased (Figure 6b). In these samples, half of the cells still retained a morphology similar to the control (star‐shaped and spread), but showed a higher density of integrin clusters with centripetal distribution in the perinuclear area. In the other half of the cells, characterized by spindle‐shaped and elongated morphology, the α5β1 clusters became point‐like, smaller, distributed in the perinuclear area, and at lateral intercellular surfaces forming cell–cell contact points (Figure 6b). After laser treatment, fibroblasts appeared similar to the controls, both for the signal intensity and distribution of α5β1 clusters (Figure 6c) 

Figure 6. Effect of laser treatment on α5β1 expression and distribution. Microscopy analysis of α5β1 expression evaluated by immunofluorescence (magnification 100×) on NHDF in basal conditions (CTRL; panel (a)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panel (b)), stimulated with IL‐1β and TNF‐α for 48 h and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + Laser; panel (c)). Bar = 10 μm. The histogram reports the mean pixel intensity, acquired by ImageJ software after appropriate thresholding and subsequent image masking (panel (d)). * p < 0.05 CYKs group vs. CTRL group; # p < 0.05 CYKs + Laser group vs. CYKs group (n = 3).

Through its interaction with different cell types, cytokines, and other ECM
molecules, and facilitating collagen fibrogenesis by scaffolding action, fibronectin plays a
preeminent role in both wound healing and scarring [35,36]. Similarly to its receptor α5β1, fibronectin significantly increased in fibroblast cultures stimulated with IL‐1β and TNF‐α, when compared to unstimulated controls, and formed a dense extracellular network of fibrils (Figure 7a,b). After laser treatment, fibronectin expression returned to the basal levels observed in control cells with evident reduction of extracellular fibrils (Figure 7c,d).   

Figure 7. Effect of laser treatment on fibronectin expression and organization. Microscopy analysis of fibronectin expression evaluated by immunofluorescence (magnification 100×) on NHDF in basal conditions (CTRL; panel (a)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panel (b)), stimulated with IL‐1β and TNF‐α for 48 h and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + Laser; panel (c)). Bar = 10 μm. The histogram reports the % of the surface area with fibers, acquired by ImageJ software after appropriate thresholding to only include the stained fibers (panel (d)). * p < 0.05 CYKs group vs. CTRL group;
# p < 0.05 CYKs + Laser group vs. CYKs group (n = 3).

In addition, the synthesis of collagen I, one of the most abundant ECM components,
was significantly enhanced by the exposure to the cytokine mix in comparison with
control unstimulated cells (Figure 8a,b). Interestingly, stimulated fibroblasts showed an
intracellular accumulation of collagen I, apparently in the endoplasmic reticulum and/or
Golgi apparatus, while the protein was not released in the extracellular environment
(Figure 8b). Cytokine‐mix stimulated fibroblasts exposed to the laser treatment revealed a collagen I signal similar to that observed in the control, both for distribution and
fluorescence intensity (Figure 8c,d).
Matrix metalloproteinases (MMPs) are endopeptidases that can degrade the ECM
proteins. They have important roles in fundamental physiological processes, such as
embryonic development, morphogenesis, and tissue remodeling, and are involved in a
number of diseases. MMPs are present in both acute and chronic wounds, where they
regulate ECM degradation/deposition that is essential for wound healing. The excess
protease activity can lead to chronic nonhealing wounds.

Figure 8. Effect of laser treatment on Collagen I expression and distribution. Microscopy analysis of Collagen I expression evaluated by immunofluorescence (magnification 100×) on NHDF in basal conditions (CTRL; panel (a)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panel (b)), stimulated with IL‐1β and TNF‐α for 48 h and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + Laser; panel (c)). Bar = 10 μm. The histogram reports the mean pixel intensity, acquired by ImageJ software after appropriate thresholding and subsequent image masking (panel (d)). * p < 0.05 CYKs group vs. CTRL group; # p < 0.05 CYKs + Laser group vs. CYKs group (n = 3).

Specifically, MMP‐1 is able to degrade collagen types I, II, and III. Similarly to
fibronectin and collagen, also MMP‐1 significantly increased in fibroblasts stimulated
with the cytokine mix (Figure 9b), compared to non‐stimulated controls (Figure 9a). Laser
treatment counteracted the effect of the cytokine mix and reported MMP‐1 expression to
a level comparable to that found in the basal state (Figure 9c).

Figure 9. Effect of laser treatment on MMP‐1 expression and distribution. Microscopy analysis of MMP‐1 expression evaluated by immunofluorescence (magnification 100×) on NHDF in basal conditions (CTRL; panel (a)), stimulated with IL‐1β and TNF‐α for 48 h (CYKs; panel (b)), stimulated with IL‐1β and TNF‐α for 48 h and then exposed to laser treatments (3 treatments, repeated once a day, for 3 consecutive days) (CYKs + Laser; panel (c)). Bar = 10 μm. The histogram reports the mean pixel intensity, acquired by ImageJ software after appropriate thresholding and subsequent image masking (panel (d)). * p < 0.05 CYKs group vs. CTRL group; # p < 0.05 CYKs + Laser group vs. CYKs group (n = 3).

4. Discussion

The cytokines IL‐1β and TNF‐α have already been used at different concentrations,
individually or in association, to stimulate an inflammatory response in various cell types,
dermal fibroblasts included [28,38,39]. In this study, IL‐1β and TNF‐α have been used
jointly to induce a pro‐inflammatory phenotype in dermal fibroblasts, with the aim to
investigate if PBMT delivered via a dual‐wavelength NIR laser system (MLS‐MiS) was
effective in counteracting cell inflammatory response and modulating fibroblast functions
involved in stromal activation, wound healing, and its alterations, which can lead to
chronic ulcers or fibrosis. Preliminary experiments performed to define the protocol for
preparing the in vitro model of inflammation in dermal fibroblast cultures showed that
both 24 h and 48 h exposure to IL‐1β and TNF‐α produced a significant increase in the
inducible enzyme mPGES‐1 and in the release of its product PGE‐2. The increase in
mPGES‐1 was higher after 48 h, while the increase in PGE‐2 was similar at 24 h and 48 h.
Therefore, a stimulation time of 48 h was chosen for the subsequent experiments in which non‐stimulated controls, samples stimulated with IL‐1β and TNF‐α, and samples exposed to laser radiation after the stimulation with the inflammatory cytokines were compared for their morphology, inflammatory profile, and expression of molecules involved in ECM remodeling. In stimulated samples, the inflammatory signals iNOS, COX‐2, and mPGES‐1 significantly increased in comparison with non‐stimulated controls, in agreement with data reported in literature [40] and supporting the validity of the inflammatory model used.

Samples stimulated and then treated with PBMT showed a significant decrease in iNOS and COX‐2, compared to the stimulated but non‐laserirradiated samples. The mPGES‐1 level and that of the final product PGE‐2 decreased, but not significantly, suggesting a multimodal action of PBMT, which could act at different cellular levels (gene transcription, protein expression, and localization), as demonstrated by the reported results.   
Modulation of the three mediators mentioned above is closely related to their
upstream activator NF‐κB, an inducible transcription factor which is activated upon
binding of pro‐inflammatory cytokines, such as TNF‐α, to their membrane receptors. In
basal conditions, NF‐κB is sequestered in the cytoplasm by a family of inhibitory proteins.
Following inflammatory stimuli, this protein moves to the nucleus, binds to specific elements on DNA, and recruits cofactors forthe transcription oftarget genes iNOS,COX‐2, andmPGES‐1. In the presence of inflammatory stimuli, the co‐localization of the transcription factor NF‐κB within the nucleus, observed by immunofluorescence, correlates with an increased
expression of iNOS, COX‐2, and mPGES‐1 at the cytoplasmic level and a consequent increase of PGE‐2 released in the extracellular medium. Following laser treatment, these values are
significantly reduced, demonstrating the effectiveness of the laser source and treatment
parameters used in counteracting the inflammatory response.
The anti‐inflammatory properties of the NIR source used had already been
highlighted by a proteomics study on laser‐irradiated myoblasts, in which a marked
increase in NLRP10, a strong inhibitor of the inflammasome, and in turn of IL‐1β and
interleukin‐18 (IL‐18) release, was observed [25]. These data are in agreement wit 
previous studies showing the effectiveness of red and NIR radiation in reducing the
inflammatory signals both in fibroblast cultures and at the wound level [42,43].   
As previously mentioned, inflammation is a protective response characterized by a
series of reactions modulated by the master regulator NF‐kB, whose gene targets are
involved both in the recruitment of immune cells to the site of injury and in vasodilation. 

Affect markers of fibroblast activation, substantiated the safety of laser irradiation on
quiescent unstimulated cells.
IL‐1β  and TNF‐α  treatment induced also noticeable morphological changes with
cytoskeletal rearrangements in the network of microtubules and actin microfilaments.
Microtubules form a scaffold which controls cell shape, intracellular transport,
signaling, and organelle positioning. Microtubules are stiff and intrinsically polarized
structures built of directionally aligned αβ‐tubulin dimers. Their “minus” end is anchored
at so called microtubule‐organizing centers, whereas the “plus” ends can extend or shrink
and interact with different intracellular structures [49]. Cells able to readily reorient their
polarity axis, such as fibroblasts, generally present a radially organized microtubule array,
whose changes are mutually related to cell polarity and can mechanically contribute to
cell asymmetry by promoting cell elongation [34]. The results of the present study show
that, following cell activation by cytokines, changes in microtubule density and
orientation occurred and probably contributed to the observed cell elongation.   
Following IL‐1β and TNF‐α stimulation, the actin filament network changed as well.
Density and thickness of actin filaments increased, while their distribution underwent a
rearrangement, giving rise to an array of filaments aligned parallel to the major cell axis.
Considering that microtubules are connected to the layer of actin filaments close to the
cell membrane through a complex of adaptor proteins often associated with focal
adhesions, the changes in microtubule and actin filament networks are probably
interrelated, and the rearrangement in α5β1 membrane integrin distribution observed in
cytokine‐stimulated cells further support this hypothesis. The inhibition of the
inflammatory response due to laser treatment led to a partial recovery of the basal
cytoskeleton organization in fibroblasts irradiated after cytokine stimulation.
Cytoskeleton changes connected with effects produced by NIR laser irradiation have been
previously described in different cell models [25,50] and depend on cell type, cell status,
parameters, and sources used.   
α‐SMA is one of the six actin isoforms. Together with β‐ and γ‐actin isoforms, α‐SMA
is expressed in some fibroblast/myofibroblast subpopulations in the basal state, where it
has a cytoplasmic localization and participates in stress fiberformation. α‐SMA is strongly
induced by mechanical stress and TGF‐β1 in activated myofibroblasts [51], therefore it is
generally considered a marker of fibroblast‐myofibroblast transdifferentiation. In the
present study, dermal fibroblasts stimulated with IL‐1β  and TNF‐α  showed  α‐SMA
expression seemingly concentrated in the nucleus, while cytoplasmic stress fibers, to some extent present in unstimulated cells, completely disappeared in the stimulated ones. 

These results are consistent with in‐depth investigations on  α‐SMA distribution and roles
carried out in the last two decades. It has been demonstrated that the apparent nuclear
localization is due to deep invaginations of the nuclear membrane filled of α‐SMA [52].
The role of the nuclear invaginations is currently quite completely unknown, but it has
been hypothesized that these structures could be involved in cellular and nuclear
mechanotransduction, nuclear transport, calcium signaling, cell differentiation [52–54].
Moreover, in agreement with our results, it has been found that TNF‐α suppresses α‐SMA
expression and stress fiber formation in dermal fibroblasts and that persistent
inflammation, mediated by TNF‐α, might prevent normal matrix deposition and
myofibroblast‐dependent wound contraction mediated by TGF‐β1 in physiological
wound healing [55]. The inhibition of stress fiber formation would turn the cells into a
phenotype more migratory and less able to generate tractional forces [51], with possible
consequences and delay in the healing process.   
Additionally, in the case of  α‐SMA, NIR laser treatment after IL‐1β  and TNF‐α 
partially prevented the cytokine effect and some stress fibers reappeared inside the cells.
α‐SMA expression following red‐ or NIR‐laser treatment has been widely studied being
connected with the effectiveness of laser therapy in promoting wound healing and
avoiding scarring. The results have been controversial, showing both down‐  and up‐
regulation of α‐SMA expression [56–58]. This variability in results is possibly due to theIn this study, fibroblast production of VEGF at the cytoplasmic level and its secretion
in the extracellular milieu were therefore analyzed. Compared to untreated controls, IL‐
1β  and TNF‐α  stimulation of fibroblasts induced an increase in VEGF production and
release in culture medium, that further confirms the validity of the model used for the
present study. The proinflammatory cytokines‐induced enhancement in VEGF levels is
widely documented in vitro [44], and it occurs in vivo in chronic inflammatory diseases
as well as in acute inflammatory response to infections and injuries. VEGF is produced by
the most part of cell populations involved in wound healing, as platelets, immune cells
(neutrophils and macrophages), fibroblasts, and endothelial cells, and reaches the
maximum concentration during the proliferative phase. In the wound, VEGF promotes
angiogenesis [45] and influences re‐epithelialization and collagen deposition through
stimulation of keratinocytes and fibroblasts [46]. However, if in a proper inflammatory
response VEGF upregulation is needed to promote angiogenesis, excessive or persistent
inflammation and VEGF production can lead to fibrosis and should be controlled. Laser
treatment subsequent to IL‐1β and TNF‐α stimulation abolished the cytokine‐mediated
VEGF increase and brought VEGF levels back to values even lower than those seen in
unstimulated controls. In literature, a modulation of VEGF expression following
irradiation with red‐NIR wavelengths has been described, the final effects depending on
irradiation parameters and experimental models used [42,47,48]. The decrease observed
in the present study, irradiating activated fibroblasts with the source and parameters
described, further supports the strong anti‐inflammatory action of the proposed laser
treatment. At the same time, the results on cells exposed to laser alone, which did not affect markers of fibroblast activation, substantiated the safety of laser irradiation on quiescent unstimulated cells.

IL‐1β and TNF‐α treatment induced also noticeable morphological changes with cytoskeletal rearrangements in the network of microtubules and actin microfilaments.
Microtubules form a scaffold which controls cell shape, intracellular transport, signaling, and organelle positioning. Microtubules are stiff and intrinsically polarized structures built of directionally aligned αβ‐tubulin dimers. Their “minus” end is anchored at so called microtubule‐organizing centers, whereas the “plus” ends can extend or shrink and interact with different intracellular structures [49]. Cells able to readily reorient their polarity axis, such as fibroblasts, generally present a radially organized microtubule array, whose changes are mutually related to cell polarity and can mechanically contribute to cell asymmetry by promoting cell elongation [34]. The results of the present study show that, following cell activation by cytokines, changes in microtubule density and orientation occurred and probably contributed to the observed cell elongation.
Following IL‐1β and TNF‐α stimulation, the actin filament network changed as well. Density and thickness of actin filaments increased, while their distribution underwent a rearrangement, giving rise to an array of filaments aligned parallel to the major cell axis. Considering that microtubules are connected to the layer of actin filaments close to the cell membrane through a complex of adaptor proteins often associated with focal adhesions, the changes in microtubule and actin filament networks are probably interrelated, and the rearrangement in α5β1 membrane integrin distribution observed in cytokine‐stimulated cells further support this hypothesis. The inhibition of the inflammatory response due to laser treatment led to a partial recovery of the basal cytoskeleton organization in fibroblasts irradiated after cytokine stimulation.

Cytoskeleton changes connected with effects produced by NIR laser irradiation have been previously described in different cell models [25,50] and depend on cell type, cell status, parameters, and sources used.
α‐SMA is one of the six actin isoforms. Together with β‐ and γ‐actin isoforms, α‐SMA is expressed in some fibroblast/myofibroblast subpopulations in the basal state, where it has a cytoplasmic localization and participates in stress fiber formation. α‐SMA is strongly induced by mechanical stress and TGF‐β1 in activated myofibroblasts [51], therefore it is generally considered a marker of fibroblast‐myofibroblast transdifferentiation. In the present study, dermal fibroblasts stimulated with IL‐1β and TNF‐α showed α‐SMA expression seemingly concentrated in the nucleus, while cytoplasmic stress fibers, to some extent present in unstimulated cells, completely disappeared in the stimulated ones. These results are consistent with in‐depth investigations on α‐SMA distribution and roles carried out in the last two decades. It has been demonstrated that the apparent nuclear localization is due to deep invaginations of the nuclear membrane filled of α‐SMA. The role of the nuclear invaginations is currently quite completely unknown, but it has been hypothesized that these structures could be involved in cellular and nuclear mechanotransduction, nuclear transport, calcium signaling, cell differentiation [52–54]. Moreover, in agreement with our results, it has been found that TNF‐α suppresses α‐SMA expression and stress fiber formation in dermal fibroblasts and that persistent inflammation, mediated by TNF‐α, might prevent normal matrix deposition and myofibroblast‐dependent wound contraction mediated by TGF‐β1 in physiological wound healing [55]. The inhibition of stress fiber formation would turn the cells into a phenotype more migratory and less able to generate tractional forces, with possible consequences and delay in the healing process.

Additionally, in the case of α‐SMA, NIR laser treatment after IL‐1β and TNF‐α partially prevented the cytokine effect and some stress fibers reappeared inside the cells. α‐SMA expression following red‐ or NIR‐laser treatment has been widely studied being connected with the effectiveness of laser therapy in promoting wound healing and avoiding scarring. The results have been controversial, showing both down‐ and up‐ regulation of α‐SMA expression. This variability in results is possibly due to the many different models (from cell cultures to animal models both normal and representing serious diseases, such as diabetes), laser sources, treatment protocols and parameters, and times at which analysis of α‐SMA expression was performed. Interestingly, some studies in which the analysis of α‐SMA expression was performed at different healing times after laser treatment showed that α‐SMA expression changed in the different healing phases and resulted significantly different from controls only at specific time points [59]. The only unambiguous result is that laser irradiation is able to modulate α‐SMA, but the modulation depends on many factors, among which the healing phase and corresponding cell phenotype (e.g., the phenotype of fibroblasts in the inflammatory phase is different from what they assume in the remodeling phase). This means that further studies are needed to develop treatment protocols suitable for the different patient’s conditions and, in case of wounds, healing phase. However, the data of the present study indisputably demonstrate that, even at the cytoskeletal level, the source and the treatment parameters used are effective in counteracting the changes induced by cytokine stimulation, thus returning the cells to the basal state.

Compared to controls, the IL‐1β and TNF‐α stimulated fibroblasts showed increased fibronectin (FN) expression and assembly observed in the same samples. The increase in FN, a major ECM component, could be expected since the pro‐inflammatory cytokines IL‐ 1β and TNF‐α, together with TGF‐β, are considered potent fibrogenic initiators [60]. The increase in expression of α5β1 observed in the same samples is consistent with that of FN, considering that α5β1 is a membrane integrin able of binding FN [61].
A number of studies investigated the expression and role of α5β1 and its ligand FN in fibroblasts during inflammation and wound healing. In the healing process, α5β1‐ mediated fibroblast‐FN interaction is crucial: α5β1 is involved in myofibroblast differentiation and granulation tissue formation by promoting FN assembly in a fibrillar structure. In the granulation tissue, a reduced ability to bind FN via integrin α5β1 might allow fibroblasts to migrate in the early FN‐rich matrix and invade the wound [62]. On the other hand, some studies demonstrated that α5β1 integrin is able to confer strong cohesivity to 3D cellular aggregates linking adjacent cells together via FN, and that the FN with its dimeric structure is essential for this process [63]. Moreover, it has been suggested that α5β1‐FN interaction contributes to clot retraction [63]. Therefore, α5β1 and FN play a crucial role in wound healing, and alterations in their expression can lead to healing impairment and fibrosis. These conditions can affect ECM remodeling by stimulating collagenase production and stimulating/inhibiting collagen/glycosaminoglycan biosynthesis depending on the target cells and experimental conditions.

The effects of the pro‐inflammatory cytokines IL‐1β and TNF‐α on ECM remodeling and their role in fibrosis have been studied for many years with controversial results. In the present study, the cytokine‐stimulated dermal fibroblasts showed increased expression of MMP‐1 and collagen I, which have key roles in ECM degradation and building, respectively, thus modulating ECM turnover. In agreement with literature, the intracellular distribution of MMP‐1 was associated with mitochondria [64] and, probably, the cytoskeleton. In fact, a relation between actin system dynamics and MMPs has been speculated because it has been observed that cytoskeleton changes often precede MMPs modulation and actin microfilament dynamics might be linked to the expression of MMP genes [65]. Regarding collagen I, contrary to what observed for FN, in stimulated fibroblasts it showed an intracellular localization and no extracellular fibrils were observed. If an increase in MMP‐1 expression following IL‐1β and/or TNF‐α stimulation has been unanimously reported [39,66–68], the effects the two cytokines have on collagen synthesis remain uncertain. Many studies reported that both IL‐1β and TNF‐α inhibit collagen I synthesis [67,69–71], but other studies demonstrated that IL‐1β and TNF‐α increased collagen I synthesis in human renal fibroblasts [72] and in murine intestinal myofibroblasts [38], respectively. A proposed scenario is that, in some conditions, the antifibrotic effect of TNF‐α is overwhelmed by its central role in driving inflammation.

Fibroblasts stimulated with IL‐1β and TNF‐α and then exposed to NIR laser radiation recovered features more similar to unstimulated controls as regards the expression and distribution of α5β1, FN, collagen I, and MMP‐1. Therefore, laser treatment was also able to counteract the cytokine effects on α5β1 integrin and the proteins involved in ECM turnover and remodeling after injury. It is noteworthy that, in the case of FN, not only the expression returned to levels comparable with unstimulated controls, but in samples treated with laser radiation the fibrils showed a more ordered and parallel distribution. This effect of laser radiation on FN and collagen fibril organization has already been described [73] and could be connected with the laser radiation’s ability to prevent fibrotic scars. The influence of red‐NIR laser radiation on the expression of α5β1 integrin, FN, collagen, and MMP‐1 has already been investigated in studies concerning laser application in the management of inflammatory response and wound healing. The results of these studies are controversial. A recent study on a model of diabetic wounded fibroblast cells showed that PBMT (660 nm wavelength) downregulated the expression of the genes FN1, ITGA5, and ITGB1, encoding for FN, α5, and β1 integrin subunits, respectively [74]. In a study on an immunosuppressed rat wounded model, PBMT by an 810 nm pulsed laser induced an increase in FN expression [75]. Enhanced FN expression was found also in human fibroblasts irradiated with a 940 nm diode laser [58]. A research on the effects of different protocols of PBMT in the healing of open wounds in rats showed that all the protocols used induced an increase in collagen deposition, but at different extent, depending on wavelength and fluence applied [43]. Using similar fluence but a different wavelength, a decrease in collagen production was found in wounded human skin fibroblasts [76]. In a rat model of wound healing, collagen deposition did not increase 3 days after laser treatment, but it increased significantly at day 7 after treatment [77]. Sakata et al. [28] found that, in chondrocytes stimulated with IL‐1β, MMP‐1 increased and then decreased after NIR irradiation applied post‐stimulation, in complete agreement with what has been observed in the present study on fibroblasts activated by IL‐1β and TNF‐α.

From the outcomes of the studies mentioned above, it is evident that expression and function of α5β1 integrin, FN, collagen, and MMP‐1 can be modulated through application of PBMT. However, results so uneven as those reported in the literature about PBMT effects demonstrate once again that it is very difficult to compare studies carried out using different experimental models, laser sources, and treatment parameters. Laser source and treatment protocol should be characterized for their biological effects before application for the management of specific pathological conditions.
In this paper, an in vitro model of fibroblast activation via stimulation with the pro‐ inflammatory cytokines IL‐1β and TNF‐α has been proposed and used to test the anti‐ inflammatory effect of a dual wavelength NIR laser source widely used in clinics to promote healing and reduce inflammation and pain. Like all in vitro models, a limit of the proposed model is to provide a very partial representation of what happens in vivo during inflammation and healing (a single cell population and two pro‐inflammatory cytokines vs. many cell populations and a plethora of pro‐ and anti‐inflammatory molecules). 

However, it can be considered representative of the early stage of the inflammation phase after an injury, when M1 macrophages produce great amounts of IL‐1β and TNF‐α. In the normal evolution of inflammation, macrophage phenotype is expected to shift from M1 to M2, with increased TGF‐β production and a decrease in IL‐1β and TNF‐α levels [60]. Therefore, the proposed model can be considered also representative of altered evolution of inflammation with persistence of high levels of TNF‐α, compared to TGF‐β levels, due to the failure to switch from M1 to M2.
Using this model, the effectiveness of PBMT by a dual wavelength NIR laser source (MLS‐MiS) in reducing inflammation has been tested, and the results obtained show that PBMT, administered through the laser source and protocol here described, is significantly effective in preventing the effects of IL‐1β and TNF‐α, thus modulating the cell inflammatory response and favoring cell return to the basal physiological state.

The anti‐inflammatory effect of red‐NIR laser radiation has been already reported in a number of studies but, to the best of our knowledge, it is the first time that it is evaluated and confirmed in an “in vitro” model of IL‐1β and TNF‐α activated dermal fibroblasts. Moreover, the significant anti‐inflammatory activity of the laser emission tested in the present research is consistent with our previous studies carried out with the same laser source. In an in vitro model of myoblasts, it was found to increase the expression of NLRP10, a potent inhibitor of inflammasome activation and IL‐1β and IL‐18 production, as well as that of PP1, which regulates many important cell functions and favors cell recovery from stress to basal state [25]. In vivo, the same laser emission was able to reduce inflammatory infiltrate and accelerate the healing of ulcers in feline stomatitis [78] while, in a rat model of neuropathic pain induced by trauma, it significantly lowered inflammation and pain and preserved the myelin sheath [79]. It is well known that, when released by cells under pro‐inflammatory stimuli, IL‐1β and IL‐18 induce the production of other pro‐inflammatory cytokines, such as interferon‐γ (INF γ), TNFα, IL‐6, etc., thus triggering a cascade of events which further increase and perpetuate inflammation. Therefore, the ability of the proposed laser treatment to inhibit IL‐1β and IL‐18 release, through increased NLRP10 production, could explain its effectiveness in controlling fibroblast activation induced by IL‐1β and TNF‐α stimulation, thus damping excessive inflammatory response. Further studies could help to define treatment protocols specific for each different healing phase.

Author Contributions: Conceptualization, M.M. and L.M.; methodology and investigation, F.C., V.C., S.G., E.S.; resources, M.M. and L.M.; writing—original draft preparation, F.C., V.C., S.G., M.M., L.M., E.S.; writing—review and editing, F.C, M.M., L.M.; supervision, project administration, and funding acquisition, M.M and L.M. All authors have read and agreed to the published version of the manuscript.

Funding: This research was partially funded by the European Space Agency (ESA) in the frame of the MAP Project “WHISPER—Wound Healing in Space: problems and PErspectives for tissue Regeneration and engineering”, SciSpacE Microgravity Application Promotion Programme, ESA Contract Number 4000130928/20/NL/PG/pt.

Instituional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement: The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Acknowledgments: The authors acknowledge ASA Srl for providing the laser source used in this study.
Conflicts of Interest: The authors declare no conflict of interest. The sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.