Journal of Photochemistry and Photobiology B: Biology€¦ · photobiomodulation through low level light/laser therapy (LLLT) ... tive for the treatment of Herpes Simplex Labialis

Journal of Photochemistry and Photobiology B: Biology xxx (2011) xxx–xxx

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Journal of Photochemistry and Photobiology B: Biology

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Enhancement of cutaneous immune response to bacterial infection afterlow-level light therapy with 1072 nm infrared light: A preliminary study

Seung Yoon Celine Lee a,b,⇑, In-Wha Seong a, Ji-Seon Kim a, Kyeong-A. Cheon a, Se Hun Gu a,Hee Hwan Kim c, Ki Ho Park d

a Department of Microbiology, Korea University Medical School, 126-1, 5-Ga, Anam-Dong, Seongbuk-Gu, Seoul 136-705, South Koreab Hayan-nara Dermatology Group, 142-3, Sankok-dong, Bupyeong-ku, Incheon, South Koreac Department of Pathology, Korea University Medical School, 126-1, 5-Ga, Anam-Dong, Seongbuk-Gu, Seoul 136-705, South Koread Quantitative Real-Time PCR Lab, Clinical Research Institute, Seoul National University Hospital, 28, Yeongun-dong, Jongno-ku, Seoul, South Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 April 2011Received in revised form 30 August 2011Accepted 30 August 2011Available online xxxx

Keywords:Photobiomodulation1072 nmLow level light therapyLEDAntibacterialVEGF

1011-1344/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2011.08.009

⇑ Corresponding author at: Hayan-nara DermatologBupyeong-ku, Incheon, South Korea. Tel.: +82 10 9991

E-mail address: [email protected] (S.Y. Celine

Please cite this article in press as: S.Y. Celine Lewith 1072 nm infrared light: A preliminary stu

We investigated the photobiomodulation effects of 1072 nm infrared light on the natural immuneresponse involved in anti-bacterial and wound healing processes. Thirty mice infected with MRSA onthe skin were divided into two groups. The experimental group was treated with 1072 nm infrared light(irradiance: 20 mW/cm2, fluence: 12 J/cm2 for 10 min) at 2, 4, 8, 12, 24 h, 3 and 5 days after inoculationand the control group with sham light. Serial changes of the mRNA levels of TLR2, IL-1b, TNF-a, IL-6, iNOS,MCP-1, TGF-b, bFGF and VEGF were studied by real time RT-PCR and those of the expression level ofVEGF, bFGF, TGF-b and NF-jB by immunohistochemistry. The mRNA levels of the cytokines involvedin the early phase of anti-bacterial immune response (IL-1b, TNF-a, IL-6, MCP-1) increased significantlyin the 1072 nm group, peaking between 12 and 24 h post-inoculation. These levels normalized after 3–5 days. Immunohistochemistry revealed a notably stronger expression of VEGF in the 1072 nm groupfrom 8-h post-inoculation to 5-day post-inoculation. We concluded that 1072 nm infrared light had aphotobiomodulation effect which resulted in an enhanced biological immune response to the bacterialinfection by MRSA and also increased the expression of VEGF to a significant level.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction tive for the treatment of Herpes Simplex Labialis (HSL), the com-

Photobiomodulation is the process where incident photons oflight are absorbed by chromophores of living tissue, for examplecytochrome C oxidase in the respiratory electron transport chainof mitochondria, which results in modulation of various cell func-tions [1–3]. It has been demonstrated by many in vitro studies thatphotobiomodulation through low level light/laser therapy (LLLT)in the waveband of 600–1000 nm can significantly stimulate cellu-lar proliferation and cellular activities in a variety of cell lines, suchas keratinocytes, epithelial cells, skeletal muscle cells, and variousdermal cells that are involved in the wound healing process includ-ing fibroblasts, macrophages, and mast cells [3–8]. Furthermore,LLLT has been proven to facilitate the chemotactic and phagocyticactivities of neutrophils and macrophages, with the latter shownto be enhanced in its synthetic activity of fibroblast growth factor(FGF) by LLLT, which resulted in increased collagen synthesis andfaster wound healing [5–8].

Among various wavelengths that have been studied for theirphotobiomodulation effects, 1072 nm has been found to be effec-

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y Group, 142-3, Sankok-dong,8406; fax: +81 31 711 8406.Lee).

e et al., Enhancement of cutandy, J. Photochem. Photobiol. B:

mon vesicular eruptive disease around the lips caused by HerpesSimplex Virus (HSV) [9,10]. In an in vitro study, this particular wave-length has been proven to have a cytoprotective effect for lympho-cytes that were exposed to insult of ultraviolet irradiation [11].Recently, a pilot study conducted in our laboratory showed that,in nude mice infected with Methicillin-resistant Staphylococcus aur-eus (MRSA), irradiation with 1072 nm infrared light shortened thetime duration for the infected wound to heal completely (unpub-lished data). We also found that direct irradiation of 1072 nm overS. aureus colonies did not show a reduction of the colony count,which suggested that this shortened healing time of the infectionmight involve participation of immune cell functions rather thana direct anti-bacterial effect of the wavelength. Herein, to investi-gate further about this LLLT action of 1072 nm of indirect antibac-terial and wound healing effect, we designed a placebo-controlledanimal study with a mouse model infected with MRSA.

2. Material and methods

2.1. Study design

Thirty female BALB/c nude miceaged 6–8 week-old were ob-tained for this study (Japan SLC, Inc., Hamamatsu, Japan). Animals

eous immune response to bacterial infection after low-level light therapyBiol. (2011), doi:10.1016/j.jphotobiol.2011.08.009

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2 S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2011) xxx–xxx

were housed separately and were maintained on a 12-h light/darkcycle with food and water. On day 1, animals were anesthetizedwith an intraperitoneal injection of Rompun (Bayer) and Zoletil50 (Laboratories Virbac). Then, a 1.5 � 2.5 cm wound area was cre-ated on the back of the mice by repeated (7–10 times) tape-strip-ping and subsequent gentle abrasion with sand paper to removethe epidermal barrier [12]. A 20 lL suspension of MRSA containing2.4 � 1010 bacteria was then applied evenly to the wound area.After applying the bacteria, all mice were randomly assigned toeither the control group (n = 15) or the experimental group(n = 15) and placed into individual cages separated by plastic bar-riers. Free access to food and water and enough space to exercisewere allowed for all subjects. However, physical contact betweenthe mice was strictly inhibited by the use of plastic walls. Beforestarting the actual experiment, 2 h were allotted for the bacterialintrusion into the skin and this time point was set as ‘baseline’.Irradiation with 1072 nm LED array over the dorsum of the subjectanimals commenced 2 h after the inoculation (baseline) and re-peated at 4, 8, 12, 24 h, 3 days and 5 days after the inoculation.The time-points for evaluation were set based on a previous publi-cation that proved the establishment of a superficial skin infectionmodel using S. aureus in a 6–8 week-old BALB/c female mice [12]and also on the results of our preliminary study which was con-ducted to see the natural course of skin infection in the samemouse model without any intervention. The early time-pointswithin the first 24 h (4, 8, 12, 24 h) were chosen to investigatethe effect of 1072 nm LLLT on the early or acute inflammatoryphase, the later time-points (3- and 5-day) on the late inflamma-tory phase and wound healing process.

Prior to each evaluation point, the experimental group received10 min of 1072 nm infrared light (bandwidth ±50 nm) from an LEDplanar array positioned approximately 3 cm above the mice. Thelight source had an irradiance of 20 mW/cm2 and fluence of 12 J/cm2 for the treatment duration of 10 min, and was equipped witha cooling fan. These parameters were almost identical to those atthe skin level of the subjects which were located at a distance of3 cm (attenuation of the irradiance less than 3%). At this level ofirradiance and fluence, with the addition of a cooling fan, heat pro-duction was too low to cause any temperature change of the sub-jects and experimental environment. The subjects of the controlgroup were treated with sham LED panels (no light) equipped withthe same cooling fans as the actual device. This ensured that allsubjects were treated under the same environment and levels ofstress.

Biopsy specimens were taken 20 min after the light treatmentat every treatment time-point. Two subjects from each group(control group and experimental group) were taken for biopsyat every time-point. Each subject was used for only one biopsyat a given time-point, not for multiple biopsies over multipletime-points, because multiple biopsies on the same animal overtime could change the wound environment and also systemic re-sponses (e.g. propagation of bacteria into deeper skin layers, pen-etration of the bacteria from the skin into the blood vessels andthe internal organs, causing systemic introduction of the organ-ism and possible sepsis). First, the animals were sacrificed by in-stant cervical dislocation to prevent any possible physiologicchange caused by the biopsy process and also to avoid inflictingunnecessary pain on the experimental animals. The skin of thewhole wound area (1.5 � 2.5 cm) was excised for specimen prep-aration. Immediately after the excision, about a 1 cm2 sized pieceof skin was fixed in phosphate-buffered formalin (4%) for furtherprocess for immunohistochemical staining, and the remainingtissue was immediately frozen with liquid nitrogen for RT-PCR.This size of the skin sample provided a sufficient specimen tissuethat could produce enough amount of RNA for high-qualityRT-2PCR.

Please cite this article in press as: S.Y. Celine Lee et al., Enhancement of cutanwith 1072 nm infrared light: A preliminary study, J. Photochem. Photobiol. B:

Biopsy samples were evaluated for difference in the expressionof Vascular endothelial growth factor (VEGF), basic fibroblastgrowth factor (bFGF), Transforming growth factor beta (TGF-b)and Nuclear Factor kappa-light-chain-enhancer of activated B cells(NF-jB) compared to the control group using immunohistochemis-try. Changes in mRNA levels were studied in Toll like receptor2(TLR2), Interleukins 1b and 6 (IL-1b, IL-6), Tumor necrosis factoralpha (TNF-a), inducible nitric oxide synthetase (iNOS), MonocyteChemotactic Protein-1 (MCP-1), TGF-b, bFGF and VEGF.

2.2. Immunohistochemistry

Immunohistochemical staining was performed using a peroxidetechnique. Formalin-fixed paraffin-embedded tissue blocks weresectioned to a thickness of 4 lm. Sections were deparaffinized for5 min three times in xylene and rehydrated for 5 min per sessionin serial-graded alcohol (100%, 95%, 80%, 70% alcohol). Antigen re-trieval was performed for VEGF, bFGF, TGF-b and NF-jB. For anti-gen retrieval, 10 mM citrate buffer (pH 6.0) was heated in apressure cooker for 10 min. The container was then cooled for20 min at room temperature. Endogenous peroxide activity wasblocked by 3% hydrogen peroxide in methanol for 10 min. Theslides were washed three times in Tris-buffered saline (TBS, pH7.6) for 5 min and incubated with a blocking solution (normal goatserum) at room temperature for 20 min. The antibodies then usedwere as follows: an anti-VEGF antibody (1:200, Lab Vision, Califor-nia, USA); an anti-bFGF antibody (1:50, Lab Vision); an anti-TGF-bantibody (1:100, Lab Vision); and an anti-NF-jB antibody (1:50,Lab Vision). The antibodies were incubated for 30 min at roomtemperature and washed three times in TBS for 5 min. Subse-quently, secondary antibody reaction was achieved with a Chem-Mate DAKO EnVision Detection Kit (Dako, Denmark) for about30 min at room temperature. After washing with TBS, the sampleswere stained with 3, 30-diaminobenzidine for chromogenic reac-tion and counter-stained with hematoxylin for 30 s.

2.3. Real time RT-PCR

Total RNA was extracted from the skin samples with a Trizol kit(Invitrogen Corp., Carlsbad, California). For synthesis of cDNA, re-verse PCR was performed with 20 lL reaction mixtures containing1 lL of 10 mM dNTP (Finnzymes Corp., Finland), 0.5 lL of M-MLVReverse transcriptase (Promega Corp. Madison, WI, USA), 4 lL ofM-MLV 5X buffer (Promega), 2 lL of random hexamer (10 lM),and 10 lL of total RNA template. Amplification was achieved bythe following cycle; 10 min at 25 �C, 60 min at 37 �C, 10 min at72 �C and finally 15 min at 4 �C; in Peltier thermal cycler PTC-200 (MJ Research Inc. USA). For real time PCR, mixtures were pre-pared with each 1.0 lL of forward and reverse primer (9 lM), 5 lLof fluorescent probe (2.5 lM), 10 lL of Taqman PCR 2X mastermixture (Perkin–Elmer, Applied Biosystens, Lincoln, CA, USA) and4 lL of PCR products. The PCR primers and probes used are listedin Table 1. Five microliters of reverse transcription reaction mix-ture was added as a PCR template. Relative quantitative real-timePCR was performed using the above reagents using an ABI Prism7000 Sequence Detection System (Perkin–Elmer Applied Biosys-tems, Lincoln, CA). The following procedure was used. After initialactivation of uracyl-N-glycosylase at 50 �C for 2 min, AmpliTaqGold (Applied Biosystems) was activated at 95 �C for 10 min. PCRconsisted of 45 amplification cycles (denaturation at 95 �C for15 s, annealing at 60 �C for 1 min, and extension at 60 �C for1 min). During PCR amplification, the amplified product amountwas monitored by continuous measurement of fluorescence. Theexpression of the genes was normalized versus a GAPDH (VIC/MGB probe, primer limited) as follows; the cycle number at whichthe transcripts of the genes were detectable (threshold cycle, Ct)



Table 1Assay IDs of the probes and primers for real timeRT-PCR.

Assay ID

TLR-2 Mm00442346_m1IL-1b Mm00434228_m1TNF-a Mm00443258_m1IL-6 Mm00446190_m1MCP-1 Mm99999056_m1iNOS Mm01309898_m1bFGF Mm01289197_m1TGF-b1 Mm00441724_m1VEGF Mm01281449_m1

S.Y. Celine Lee et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2011) xxx–xxx 3

was normalized against the Ct of GAPDH, which is referred to asdelta Ct. The expression of the genes relative to a reference was ex-pressed as 2�deltadeltaCt, where deltadeltaCt refers to the differencein the values of deltaCt between the test group and the reference(control).

3. Results

3.1. Immunohistochemistry

The most significant difference between the control and theexperimental groups was observed in the expression level of VEGF.In the 1072 nm treatment group, the VEGF expression started toappear remarkably strong from 8 h after the inoculation, whichwas significantly stronger than the same time-point result of thecontrol group (Fig. 1A). The location of this strong VEGF expressionwas mostly distributed in the papillary dermis and upper reticulardermis, although it was observed intensively in the deep dermis aswell 5-day post-inoculation. Conversely, no remarkable expressionof VEGF was found in the control group after 5 days.

The expression of bFGF was not significantly different betweenthe control and experimental groups until 3 days after the inocula-tion. However, after 5 days from inoculation, the bFGF expressionin the 1072 nm group appeared notably stronger than the controlgroup, distributing in the papillary dermis and the upper reticulardermis, the former being visibly denser than the latter (Fig. 1B).

With regards to the expression of TGF-b, we could not observeany meaningful expression during the first 24 h. However, moder-ate expression was observed on day 3 in the control group and onday 5 in the experimental group, mostly in the papillary dermis inboth groups (Fig. 1C).

Nuclear translocation of NF-jB represents the activation andinitiation of transcription genes that are involved in the rapid re-sponse to bacterial infection. Nuclear translocation of NF-jB wasmarkedly observed as early as 12 h after the inoculation in the1072 nm group, whereas this feature was not found at this time-point in the control group. In the control group, the nuclear tran-scription was observed at day 3 (Fig. 1D).

3.2. Real time RT-PCR (Fig. 2)

The results of real time RT-PCR, which showed the mRNA levelsof the measured cytokines and growth factors, and hence their le-vel of transcription, presented a relatively consistent tendency(Fig. 2). In the 1072 nm treatment group, the cytokines and growthfactors relating to the innate immune response to infection in-creased significantly compared to the control group between 4-hpost-treatment and 3-day post-treatment, and then started to nor-malize to the level of the control group’s measurements between3- and 5-day post-treatment.

The level of TLR2 mRNA started to increase 12 h after inocula-tion. In the 1072 nm group, its level was four times higher than


that of the control group after 12 h post-inoculation, and 11 timeshigher after 24 h post-inoculation. It decreased back to the level ofthat of the control group at day 3.

IL-1b showed more sustained increasing tendency than TLR2. ItsmRNA level was twice as high as that of the control group after12 h from inoculation. It further increased as high as 23 times, 27times and 22 times compared to the control group after 24 h,3 days, and 5 days respectively after the inoculation.

TNF-a, another primary cytokine along with IL-1b, also showeda remarkable increases starting from as early as 4 h post-inocula-tion compared to the control. The mRNA levels of TNF-a had con-tinued to increase, peaking at 24 h after the inoculation (26 timeshigher compared to the control) and normalized again to the con-trol level at 3 days after the inoculation (1.2 times compared to thecontrol).

IL-6, which is known to increase in the acute phase of inflam-mation, also showed an enhanced response compared to the con-trol. It was three times as high as the control at 1 h post-inoculation and increased up to 17 times compared to the controlafter 12 h post-inoculation. IL-6 mRNA level was normalized to thecontrol level at 24-h after the inoculation (1.9 times of the level ofthe control).

MCP-1 is known as a chemoattractant that recruits monocytesto the site of infection and helps their traffic across endothelial bar-rier. In the 1072 nm group, the mRNA of MCP-1 was markedly in-creased at 12 h from the inoculation which was nine times higherthan that of the control group. The level continued to be higherthan the control group until 3 days after the inoculation (5.8 timesat 24 h, 2 times at 3 days). The level dropped again at 5 days afterthe inoculation.

iNOS, the inducible form of nitric oxide synthase, startedincreasing from 24 h from inoculation. It was 1.7 times as high asthe control group at 24 h and 3 days after the inoculation and fur-ther increased to 2.7 times after 5 days.

The mRNA levels of VEGF were lower in the treatment groupthan the control group for the first 4 h after the inoculation. How-ever, it started increasing from 12-h post-inoculation, and in-creases as high as 14 times higher than the control at 24-h afterthe inoculation were seen. VEGF levels decreased again to the con-trol level at 3 and 5 days after the inoculation, the level of whichwas 1.6 times and 1.3 times compared to the control.

The change of the mRNA levels of TGF-b showed a similar pat-tern as that of VEGF. The levels were lower than the control duringthe first 4 h, and started increasing from 12-h post-inoculation (1.9times compared to the control) until it peaked at 24-h post-inocu-lation whose level was 14 times as high as that of the controlgroup. It decreased again to 5.8 times compared to the control after3 days and further decreased to 1.7 times 5-day post-inoculation.

bFGF showed a somewhat different pattern than othercytokines or growth factors. It was not detected for the first 12 h.The level in the experimental group remained relatively similarto those of the control group (0.83 times and 1.775 times com-pared to the control after 24 h and 3 days from the inoculation,respectively).

4. Discussion

Even though we now understand that low-level light can affectvarious biological processes after absorption of photons of light bycells [1–3], the exact roles of which specific wavelengths can affectwhich biological responses has yet to be fully elucidated. The mostwidely accepted example of this connection is that wavelengthsaround the red and near-infrared waveband can enhance thewound healing process through photobiomodulation [2,6–8]. Pho-tobiomodulation is the process where incident photons are ab-



Fig. 1. Serial change of VEGF, TGF-b, bFGF and NF-jB in immunohistochemistry.


sorbed by chromophores, for example cytochrome C oxidase in therespiratory electron transport chain of mitochondria, to modulatevarious cell functions, and is believed to result in new collagensynthesis to exert the effects leading to the healing of the wound[1–3]. Other examples are photodynamic reaction, where the pho-


tons react with a photosensitizer, either exogenous or endogenous,that leads to the creation of singlet oxygen and free radicals whichin turn destroy the cellular structures [13]. Blue light around415 nm is known to provoke the photodynamic reaction inPropionibacterium acnes (P. acnes), the causative bacteria of acne



Fig. 2. Serial changes in mRNA levels of the cytokines that are expressed in bacterial infection.


vulgaris, by reacting with the bacterial coproporphyrin III andprotoporphyrin IX that acts as the endogenous photosensitizer[14–16]. Photodynamic reaction with exogenously administeredphotosensitizer is also considered as a new method to treat localinfection including acne vulgaris [17–19] and some neoplastic con-ditions such as basal cell carcinoma [20–22]. Until now, most stud-ies that investigated the possibilities of utilizing the light-tissueinteraction for infection were focused on destruction of the tissuesby photothermolysis or photodynamic reaction [23–25]. Our study,on the other hand, was designed to investigate how low-level lightcan affect the natural anti-bacterial response of the host. We usedthe wavelength of 1072 nm, which is within the infrared range andis known to be effective for shortening the duration of herpes sim-plex infection of the lips [9,10]. The results of our study revealedthat low level light at this wavelength was able to influence thebiological processes of the host against the bacterial infection.

The most prominent changes can be summarized as two points;(1) enhanced response of primary cytokines activated againstinfection and (2) significant increases of VEGF compared to thecontrol. As seen in Fig. 2, the most significant increase in primarycytokines was observed with IL-1b, TNF-a and IL-6. These primarycytokines that act as the first responders to the infection are acti-vated immediately after the infection is detected by local immunecells such as Langerhans cells [26]. IL-6 is also an important medi-ator of the acute phase response that can be secreted by macro-phages to specific microbial molecules that are detected byreceptors of the host cells including toll-like receptor [27]. Activa-tion of these primary cytokines stimulates other immune cells tocommence anti-bacterial actions such as production of antibodiesand phagocytosis of the pathogens, and also to produce secondarycytokines and growth factors [28]. Therefore, increases in theseprimary cytokines with the treatment of 1072 nm may suggestthat the natural immune response against the infection wasboosted by photobiomodulation effect of this wavelength. Onemore aspect that should be noted is the pattern in which thesecytokines normalized to the level of the control in the later phases.


The mRNA levels of the cytokines appeared to peak around 12–24 h from the inoculation and then decreased to almost the samelevel as the control group at 3-day post-inoculation. The exceptionwas IL-1b, which started decreasing from 3 days after the inocula-tion but was not down to the control level until 5 days after theinoculation. This homeostasis is important to note, because sus-tained increases in cytokines may lead to an overreacting immuneresponse, which can be harmful to the host. The increase of TGB-b,which activates regulatory T-cells, and the increase of IL-6, whichexerts not only a pro-inflammatory action in the acute phase butalso has an anti-inflammatory effect by inhibiting IL-1b and TNF-a, may have a role in normalizing the increased mRNA levels ofthese cytokines to those of the control group [29]. MCP-1, the re-cruiter of monocytes which transform to macrophages after trans-locating to the site of infection [30], showed similar pattern asother primary cytokines.

The significant increase of VEGF was one of the most prominentdifferences of the experimental group compared to the controlgroup. VEGF is the key protein that stimulates the productionand the growth of new blood vessels when blood circulation isnot sufficient to supply enough oxygen. VEGF is known to increasein wound healing after injury or in local infections to provide moreoxygen, nutrients and immune cells that are needed for the anti-bacterial actions and wound healing processes. VEGF also acts asa chemoattractant for macrophages and granulocytes and as avasodilator with the involvement of nitric oxide [31–36]. These ef-fects of VEGF help the immune system to combat the infection andheal the wound site so that the body system can restore thehomeostasis. In the 1072 nm light treatment group, the immuno-histochemistry showed a remarkably stronger expression of VEGFfrom 8-h post-inoculation compared to the control group. This ten-dency continued through the last point of measurement, whichwas 5 days after the inoculation. In the earlier time-points, thestrong expression was observed mainly in the papillary dermisand the superficial to mid-dermis, however, in the 5-day post-inoc-ulation pictures, it was observed even in the deep dermis and the




subcutis layer. Although this study was not designed for comparingthe wound sizes between the experimental and the control groups,the wound status of these two groups were visibly different. Thewounds in the experimental group showed a markedly more ad-vanced reepithelialization and smaller area of erythema and activeinflammation than the control (Fig. 3). In the 1072 nm treatmentgroup, at 5-day post-inoculation, the sanguineous crusts had al-most fully detached leaving reepithelialized skin beneath. The re-sults of immunohistochemistry corresponded with those of realtime RT-PCR, where the mRNA levels of VEGF started exceedingthose of the control group from 12 h from the infection and in-creased up to 14 times higher than the control group at 24-hpost-inoculation. Since mRNA is synthesized when there is a great-er need for the coded proteins than pre-existing ones, the increaseof mRNA of VEGF in the 1072 nm group may mean that the1072 nm infrared treatment might have enhanced the productionof the protein VEGF, which was confirmed by the increased immu-noreactivity of the VEGF protein in the immunohistochemistry fea-tures. This is an important finding because it suggests that1072 nm infrared light may be beneficial for wound healing, espe-cially for those that lacks sufficient blood supply such as chroniculcers seen in diabetes mellitus or venous stasis.

iNOS, the inducible form of nitric oxide synthase, is believed tobe activated in the presence of inflammation or infection and toplay an important role in the host immune defense against patho-gens. The induction of iNOS usually happens in an oxidative envi-ronment, and hence, the resultant synthesis of nitric oxide leads tothe creation of superoxide products and cell toxicity, which con-tributes to the anti-bacterial response of the host [37–40]. In thetreatment group, the mRNA level was increased by up to 17 timeshigher than the control group. This result corresponds with thefinding of a previous study that investigated the photobiologicalaction of 1072 nm infrared light in in vitro environment [11]. Inthis study, the authors found that, when pre-treated with1072 nm infrared light, human peripheral blood mononuclear cells(PBMCs) produced a 4.9-fold increase in iNOS compared to the con-trol, which was measured by quantitative immunoblotting. Theyalso found that the pre-treated PBMCs showed a higher survival

Fig. 3. Clinical photos of the infected wound at 5-day post-inoculation of MRSA. The wo(B) compared to that of the control group (A). The crust on the wound of 1072 nm groupadherent to the skin (pictures in the small boxes). (The crusts were removed for clinical pduring the process of cervical dislocation.)


rate after exposure to ultraviolet irradiation. The authors hypothe-sized that increased iNOS might lead to greater production of nitricoxide, which is known as a potent inhibitor of apoptosis in variouscell types [41–43], and this increased nitric oxide may have ex-erted a cytoprotective effect for the PBMCs. Although the exactmechanism of how 1072 nm increases the amount of iNOS is notyet known, our study results can be considered as supporting evi-dence of their findings, showing that the mRNA of iNOS is in-creased by 1072 nm.

Low-level light therapy with 1072 nm infrared light has beenproven beneficial for the treatment of herpes simplex labialis,shortening the duration of the disease [9,10]. The mechanism of ac-tion by which 1072 nm infrared light effects the healing time ofherpes simplex labialis is not fully elucidated. Hypothetically, thefact that 1072 nm light treatment can elicit a cytoprotective effectfor lymphocytes, which play the major role in combating viral dis-eases, can be considered as one of the possible underlying mecha-nisms. The response to a bacterial infection, in contrast, issomewhat different than that to a viral infection, in the fact thatit relies on both innate and adaptive immune components [26].In our study results, 1072 nm infrared light treatment demon-strated the established classic effects of low-level light therapyon the innate immunity. These classic effects are exerted throughphotobiomodultion where the photons of light absorbed by themitochondria or other subcellular organelles increase the produc-tion of adenosine triphosphate (ATP), reactive oxygen species(ROS) and nitric oxide (NO), which facilitates the gene transcrip-tion of the given cells (e.g. leukocytes, macrophages, endothelialcells, fibroblasts etc.) through NK-jB and activator protein-1 (AP-1) signaling pathways [1–3]. As a result, these cells become acti-vated and enhanced in producing their responsible proteins or per-forming their given roles; for example, the lymphocytes secreteantibodies, fibroblasts produce collagen and extracellular matrix,mast cells degranulate, and macrophages increase phagocyticactivities. Our study results showed that 1072 nm infrared lighttreatment increased the mRNA levels of primary cytokines in-volved in the innate immune response and also increased theexpression of the growth factors that participate in the wound

und treated with 1072 nm LLLT showed a significantly advanced reepithelializationwas almost fully detached at the time of biopsy, while that of control group was stillhotography. In figure (A), the weak skin of the upper margin of the wound was torn




healing process. These finding suggest that low-level light therapyat the wavelength of 1072 nm may have photobiomodulation ef-fects that are beneficial for anti-bacterial immune activities andalso for the healing of the wound. One limitation of this study isthat the number of subjects biopsied at each time-point was small.Further studies employing larger number of subjects is warrantedto further investigate the photobiomodulation effect of 1072 nminfrared light.

5. Conclusion

Our study results revealed that the low-level light treatment at1072 nm can enhance the natural immune response against theskin infection with MRSA by increasing the primary cytokines thatare activated promptly after the bacterial intrusion. Most of the in-crease of these cytokines normalized to the level of the controlgroup after 3 days from the inoculation, suggesting that the highlevels of cytokines did not last longer than required, which isimportant for maintaining the homeostasis of the immune re-sponse. Our study results also showed that 1072 nm infrared lightalso increased the major growth factors involved in the woundhealing process, especially VEGF. These findings suggest that1072 nm low-level light treatment may be beneficial for improvingwounds that are prone to secondary infection and lack sufficientblood supply such as diabetic foot ulcer, stasis ulcers or pressuresores. Further investigation of the exact mechanism of actions of1072 nm low-level light therapy and optimization of the protocolsare warranted for possible use of this infrared light for the treat-ment of local infections and for management of wounds.

Acknowledgements

This study was funded by Pacer therapeutics Ltd., Pangbourne,United Kingdom, for the costs of the subject animals, immunohis-tochemical staining and real time RT-PCR processes. The 1072 nminfrared LED devices were also provided by Pacer therapeutics Ltd.All remuneration received to conduct the research was in compen-sation for time spent doing the research and was not dependentupon the results achieved.

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