Journal of Photochemistry and Photobiology B: Biology 121 (2013) 86–93

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Journal of Photochemistry and Photobiology B: Biology

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Potentiated anti-inflammatory effect of combined 780 nm and 660 nm lowlevel laser therapy on the experimental laryngitis

Renata R. Marinho a, Renata M. Matos a, Jandson S. Santos a, Maria A.G. Ribeiro b, Salete Smaniotto c,Emiliano O. Barreto c, Ronaldo A. Ribeiro d, Roberto C.P. Lima Jr. d, Ricardo L.C. Albuquerque Jr. e,Sara M. Thomazzi a,⇑a Departamento de Fisiologia, Universidade Federal de Sergipe, Av. Marechal Rondon, São Cristóvão, Sergipe, Brazilb Departamento de Odontologia, Universidade Federal de Sergipe, Av. Marechal Rondon, São Cristóvão, Sergipe, Brazilc Núcleo de Pesquisa Multidisciplinar, Universidade Federal de Alagoas, Av. Lourival Melo Mota, Maceió, Alagoas, Brazild Faculdade de Medicina, Universidade Federal do Ceará, Rua Cel. Nunes de Melo, 1315, Fortaleza, Ceará, Brazile Instituto de Tecnologia e Pesquisa – ITP, Universidade Tiradentes, Av. Murilo Dantas, 300, Aracaju, Sergipe, Brazil

a r t i c l e i n f o

Article history:Received 27 September 2012Received in revised form 6 February 2013Accepted 8 February 2013Available online 28 February 2013

Keywords:LaryngitisLow level laser therapyNeurogenic inflammation

1011-1344/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2013.02.012

⇑ Corresponding author. Address: Departamento deBiológicas e da Saúde, Universidade Federal de SergiSE, Brazil. Tel.: +55 79 21056640; fax: +55 79 210564

E-mail address: sthomazzi@gmail.com (S.M. Thom

a b s t r a c t

Reflux laryngitis is a common clinic complication of nasogastric intubation (NSGI). Since there is noreport concerning the effects of low level laser therapy (LLLT) on reflux laryngitis, this study aimed toanalyze the protective effect of single and combined therapies with low level laser at the doses of 2.1 Jand 2.1 + 1.2 J with a total irradiation time of 30 s and 30 + 30 s, respectively, on a model of neurogenicreflux laryngitis. NSGI was performed in Wistar rats, assigned into groups: NGI (no treatment), NLT17.5(single therapy), and NLT17.5/10.0 (combined therapy, applied sequentially). Additional non-intubatedand non-irradiated rats were use as controls (CTR). Myeloperoxidase (MPO) activity was assessed by col-orimetric method after the intubation period (on days 1, 3, 5, and 7), whereas paraffin-embedded laryn-geal specimens were used to carry out histopathological analysis of the inflammatory response,granulation tissue, and collagen deposition 7 days after NSGI. Significant reduction in MPO activity(p < 0.05) and in the severity of the inflammatory response (p < 0.05), and improvement in the granula-tion tissue (p < 0.05) was observed in NLT17.5/10.0 group. Mast cells count was significantly decreased inNGI and NLT17.5 groups (p < 0.001), whereas no difference was observed between NLT17.5/10.0 and CTRgroups (p > 0.05). NLT17.5/10.0 group also showed better collagenization pattern, in comparison to NGIand NLT17.5 groups. This study suggests that the combined therapy successfully modulated the inflam-matory response and collagenization in experimental model of NSGI-induced neurogenic laryngitis.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Nasogastric intubation (NSGI) is often used in clinical practice,during intensive care settings, emergency department, hospitalwards, and even in home care [1]. However, several complicationsresultant of NSGI, such as laryngopharyngeal reflux disease andaspiration pneumonia, have been reported [1,2].

Laryngopharyngeal reflux disease is defined as retrograde refluxof gastric contents up to the laryngeal and pharyngeal level beyondthe esophagus. Such disease potentially increase the risk of chroniccough, laryngitis, asthma, chronic obstructive pulmonary disease,recurrent pneumonia, hoarseness, difficulty swallowing, and laryn-geal cancer [3].

ll rights reserved.

Fisiologia, Centro de Ciênciaspe, 49100-000 São Cristóvão,74.azzi).

Treatment for laryngitis depends on the pathogen or etiology,age, clinical features, and vocal demands of the individual. It canbe treated with antibiotics, anti-inflammatory drugs (NSAID), oraland inhaled corticosteroids, proton pump inhibitors, antihista-mines, and others [4]. However, current therapy of laryngitismay fail due to adverse aerodigestive side effects of drugs suchas NSAID and corticosteroids, potentiating tissue injury during re-flux laryngitis [5].

Despite laryngitis frequency in clinical practice, there are only afew experimental studies concerning the mechanisms underlyingthe laryngeal injury secondary to nasogastric intubation [2,6].

Technological advances led to the development of new equip-ment commonly used for diagnostic and therapeutic purposes,including lasers. Lasers are sources of electromagnetic radiationor light, having special properties that are different from othersources. The low level laser therapy (LLLT) has been used for thetreatment of inflammatory diseases, with no remarkable sideeffects [7].

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Previous studies have suggested that LLLT can reduce the acutephase of the inflammatory response [8,9] and accelerate tissue re-pair in tendon and muscle injuries [9,10]. In addition, it has alsobeen reported that LLLT can reduce carrageenan-induced pawedema, the expression of cyclo-oxygenase (COX)-2 and pro-inflam-matory mediators, for instance tumor necrosis factor (TNF)-a,interleukin (IL)-1b, and IL-6, in rats [11,12]. Other studies alsodemonstrate that LLLT alters the activity of inducible nitric oxidesynthase (iNOS) [13] and stimulate the production of growthfactors involved in the healing process, which includes platelet-derived growth factor (PDGF) and transforming growth factor(TGF)-b [8].

However, to the best of our knowledge, there are no experimen-tal studies evaluating the use of low level laser in laryngitissecondary to nasogastric intubation. Therefore, this study aimedto investigate the effect of LLLT on the inflammatory process in amodel of neurogenic reflux laryngitis.

2. Materials and methods

2.1. Animals

Male Wistar rats (350–450 g) were obtained from the CentralBiotery of the Federal University of Sergipe (São Cristóvão, Brazil).Animals were randomly assigned into groups and maintained inplastic cages at controlled room temperature (21 ± 2 �C) with freeaccess to food and water, under a 12 h light/dark cycle. All theexperimental procedures were carried out during the light periodof the day (08:00 a.m. to 05:00 p.m.) and complied with the guide-lines on animal care of the Federal University of Sergipe EthicsCommittee for Animal Use in Research (CEPA/UFS 34/11), whichwas conducted in accordance with the internationally acceptedprinciples for laboratory animal use and care.

2.2. Experimental groups

Rats were randomly separated into four groups (n = 6/group):CTR – non-intubated rats; NGI – intubated rats; NLT17.5 – intu-bated rats treated with 17.5 J/cm2 laser irradiation; and NLT17.5/10.0 – intubated rats treated with 17.5/10.0 J/cm2 laser irradiation.CTR and NGI groups received simulated laser irradiation with thedevice turned off, in order to simulate the same stress in all theexperimental groups. All the animals were submitted to subcuta-neous administration of buprenorphine (0.05 mg/kg, 8/8 h, duringthe first 48 h) in order to minimize the pain.

Table 1Description of low level laser therapy parameters applied to the irradiated groups.

Parameters NLT17.5 NLT17.5/10.0

Emission Continuous mode Continuous modeWavelength (nm) 780 ± 10 780 ± 10 + 660 ± 10a

Light emitter active medium GaAlAs GaAlAs + InGaAlPPower output 70 mW 70 mW + 40 mWSpot size 0.04 cm2 0.04 cm2

Power density 1.75 W/cm2 1.75 W/cm2 + 1.00 W/cm2

Energy density 17.5 J/cm2 17.5 J/cm2 + 10.0 J/cm2

Irradiation time (each point) 10 s 10 s + 10 sTotal irradiation time 30 s 30 s + 30 sTotal energy 2.1 J 2.1 J + 1.2 JBeam divergence perpendicular

to the junction17� 17�

Tip angle 50� 50�

a The two wavelengths were applied sequentially.

2.3. Laryngitis model

For the induction of laryngitis, the animals were lightly anes-thetized with isoflurane inhalation (1.5%, generated by a calibratedvaporizer) to allow swallowing reflex and were submitted to thenasogastric intubation procedure. Therefore, the distance fromthe nose to the stomach of each animal was measured in orderto assess the mean length of the nasogastric tubes. Subsequently,a 10–13 cm narrow-bore nasogastric aspiration tube used for naso-gastric intubation in premature newborns (Mark Med� no. 4) wasinserted through the nasopharynx until it reached the stomach.The external tip was sutured to the nasal lateral cartilages (intuba-tion, day 0) [2]. At the end of the intubation period (on days 1, 3, 5,and 7), the animals were anesthetized and euthanized with halo-thane (3%, inhaled), and perfused (NaCl 0.9% w/v containing hepa-rin 0.1% v/v) through the ascending aorta. After confirmation thatthe nasogastric tube was still inserted in the stomach, the larynxwas removed.

2.4. Low level laser therapy (LLLT) procedure

All the LLLT experimental procedures were carried out using apreviously calibrated semi-conductor diode laser GaAlAs –780 nm and InGaAlP – 660 nm (Twin Laser�, MMOptics, São Paulo,Brazil). The animals were submitted to laser transcutaneous irradi-ation by perpendicular contact to the region of the larynx. Laser ar-rays were applied in three points (22, 27, and 32 mm distant fromthe jaw) based on the preliminary findings, so that whole area ofthe larynx was covered by irradiation. The larynx region of allthe animals was previously shaved, to provide better contact andabsorption of laser light. LLLT-treated groups had a total of fourirradiations sessions with 48 h interval between each laser applica-tion. The first session was performed 24 h after nasogastric intuba-tion. The parameters of LLLT are described in Table 1.

2.5. Measurement of myeloperoxidase (MPO) activity

Additionally, MPO activity, a marker of neutrophil accumula-tion, was measured in the larynx, based on the method originallydescribed by Bradley et al. [14]. Briefly, after the intubation period(1st, 3rd, 5th, and 7th day, 8 h after LLLT) the larynx was collected,weighed and homogenized (10 mg/mL) in phosphate buffer(50 mM, pH 6.0) containing 0.5% hexadecyl-trimethylammoniumbromide. The homogenates were incubated (2 h at 60 �C) to inacti-vate endogenous catalases. The supernatants were mixed with o-dianisidine dihydrochloride (0.167 mg/mL, in 50 mM phosphatebuffer) solution containing H2O2 (0.005%). Changes in absorbanceof the resulting chromophore were read at 460 nm (LabsystemMultiskan, Helsinki, Finland). The results were expressed as unitsof MPO (UMPO)/mg tissue, where one UMPO is defined as theamount of enzyme that degrades 1 lmol of H2O2/min.

2.6. Histopathological procedures

The larynxes collected at day 7 were formalin-fixed and paraf-fin-embedded according to the routine laboratorial techniques.Subsequently, serial 5 lm thick sections were obtained and stainedin hematoxylin–eosin, sirius red, and toluidine blue.

2.6.1. Assessment of inflammatory indexHistopathological sections stained in hematoxylin–eosin (HE)

were used to the descriptive analysis of the inflammatory index.The intensity of the inflammatory response was assessed as fol-lows: 1 (inflammatory cells representing less than 10% of the cellpopulation observed within the wound area), 2 (inflammatory cellsrepresenting between 10% and 50% of the cell population observedwithin the wound area), and 3 (inflammatory cells representing

Table 2The effect of LLLT on intensity of the inflammatory response after 7 days.

Intensity of the inflammation

NGI NLT17.5 NLT17.5/10.0

Rat 1 3 3 2Rat 2 3 3 2Rat 3 3 3 3Rat 4 3 3 2Rat 5 3 3 3Rat 6 3 3 2Median (range) 3 (3–3) 3 (3–3) 2 (2–3)*

Rats were separated in groups: NGI – intubated rats; NLT17.5 – intubated ratstreated with 17.5 J/cm2 laser irradiation; and NLT17.5/10.0 – intubated rats treatedwith 17.5/10.0 J/cm2 laser irradiation. 1 – inflammatory cells representing less than10% of the cell population, 2 – inflammatory cells representing between 10% and50% of the cell population, and 3 – inflammatory cells representing more than 50%of the cell population. Statistical analysis by Kruskal–Wallis test, followed byDunn’s test.* p < 0.05 vs. NGI and NLT17.5 groups.

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more than 50% of the cell population observed within the woundarea) according to Nunes et al. [15].

2.6.2. Assessment of collagen depositionHistopathological sections stained in picrosirius and analyzed

under polarized light were used to the descriptive analysis of thecollagen deposition. Collagen fibers were analyzed according totheir birefringence pattern (greenish/yellow-greenish or orange,orange-reddish), morphological appearance (wavy or stretched,thin or thick, short or long), and disposition (reticularly arrangedor interlaced).

2.6.3. Mucosal mast cells countFor mucosal mast cells count, histopathological sections were

stained with 0.5% toluidine blue (pH 0.5) [16].The histological sections were observed in a light microscope

(Zeiss, AXIOLAB), where metachromatic mast cells were stainedin violet. To count mast cells, 10 high-power fields (200�magnifi-cation) were selected, and the average number of metachromaticcells in the injured area in each group was obtained by addingthe number observed in each animal and dividing the result by 6(number of animals per group).

2.7. Statistical analysis

Statistical significant difference was verified by analysis ofvariance (one-way ANOVA) and Bonferroni’s test. The analysis ofthe inflammatory infiltrated was carried out by Kruskal–Wallistest, followed by Dunn’s test. Each time point was analyzedseparately, and two-tailed a-level of p < 0.05 was regarded assignificant.

3. Results

NGI group showed a significant increase on MPO activity, com-pared with CTR group (p < 0.001) at all days of experiment (Fig. 1).The application of 17.5 J/cm2 laser irradiation did not inhibitedMPO activity in relation to NGI group. However, a significant de-crease was observed on NLT17.5/10.0 group (p < 0.05 at 1st, 3rd,5th, and 7th day) vs. NGI group. No significant differences werefound in MPO activity in rats treated with 17.5 and 17.5/10.0 J/cm2 laser irradiation.

As presented in Table 2, the intensity of the inflammatory re-sponse was significantly decreased in NLT17.5/10.0 group com-pared to NGI and NLT17.5 groups (p < 0.05). Furthermore, asshown in Fig. 2, the granulation tissue was also found to be signif-icantly increased in NLT17.5/10.0 (p < 0.05).

Besides, the analysis of the histopathological sections (Fig. 3) re-vealed distinct patterns of morphological changes in the damagedtissues of different groups. In CTR group, no inflammatory reactionwas observed, whereas in NGI group, we found extensive

Fig. 1. The effect of LLLT on MPO activity after intubation period (1st, 3rd, 5th, and 7measured: CTR – non-intubated rats; NGI – intubated rats; NLT17.5 – intubated rats trea17.5/10.0 J/cm2 laser irradiation. All values are expressed as mean ± SEM. Statistical ana

ulcerative areas in the esophageous epithelial lining, recoveredfor fibrinous exudate. In the adjacent connective tissue, intenseinflammatory reaction comprised by neutrophils and lymphocyteswas observed infiltrating the laryngeal tissues, and disorganizingglandular structures and mucosal-associated lymphoid tissue.

Though a neutrophil infiltrate was observed in NLT17.5 group, itwas limited to the areas adjacent to the esophageous ulcer. In addi-tion, immature granulation tissue as observed in laryngeal tissuesof NLT17.5 group, represented by intense proliferation of spindle-shaped stromal cells (interpreted as fibroblasts and endothelialcells) and formation of small irregular capillaries blood vessels.On the other hand, NLT17.5/10.0 group showed intense inflamma-tory infiltrate extensively composed of lymphocytes, in associationto well-developed granulation tissue, with no ulcerative areas inthe esophageal squamous epithelium.

In this study, mast cells were identified by their round-shapedmorphology and metachromatic properties when stained withtoluidine blue (Fig. 4). Fig. 5 presents the mean number of mastcells per histological field (200� magnification) in the experimen-tal groups. Mast cells count was significantly higher in CTR groupin relation to NGI and NLT17.5 groups (p < 0.001), but there wasno difference compared to NLT17.5/10.0 group (p > 0.05). Thesecells were heterogeneously distributed throughout the normal lar-yngeal connective tissue, particularly among the mucous glands.However, in the intubated groups, mast cells were predominantlyarranged in small clusters in the margins of the injured laryngealtissue, and more rarely within the inflamed areas, irrespective tothe LLLT dose.

The analysis of the collagen deposition indicated that in NGIgroup, there was substantial loss of collagen in the esophageousconnective tissue adjacent to the ulcer zone, whereas in the laryn-geal tissues, there was deposition of slightly reticular-arranged

th day). Rats were separated in groups and mieloperoxidase activity (UMPO) wasted with 17.5 J/cm2 laser irradiation; and NLT17.5/10.0 – intubated rats treated withlysis by ANOVA followed by Bonferroni’s test. �p < 0.05 vs. NGI group (n = 6/group).

Fig. 2. The effect of LLLT on intensity of the granulation tissue after 7 days. Ratswere separated in groups: NGI – intubated rats; NLT17.5 – intubated rats treatedwith 17.5 J/cm2 laser irradiation; and NLT17.5/10.0 – intubated rats treated with17.5/10.0 J/cm2 laser irradiation. All values are expressed as mean ± SEM. Statisticalanalysis by ANOVA followed by Bonferroni’s test. �p < 0.05 vs. NGI group (n = 6/group).

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thin and delicate type III collagen fibrils. Similar pattern was seenin NLT17.5 group, although the birefringence of the newly-formedfibrils in the larynx was consistent with type I collagen. In NLT17.5/10.0 group, there was intense deposition of longer and thickerfibers of both type I and type III collagen, organized in a clearreticular disposition in both esophageous and laryngeal connectivetissues (Fig. 6).

4. Discussion

In this study, the laryngeal inflammation in rodents wasperformed according to the experimental model described byLima-Rodrigues et al. [2]. In addition, this is the first reportassessing the effects of LLLT on nasogastric intubation-inducedneurogenic laryngitis. Modulatory effects of LLLT over the acuteinflammatory response has been previously reported [17,18], and

Fig. 3. HE stained histological sections of the laryngeal specimens (three histological sefibers and fibroblasts in the connective tissue; (b) NGI – intubated rats. Neutrophils arLymphocytes and neutrophils are highlighted; and (d) NLT17.5/10.0 – intubated ratsextending to the laryngeal tissues (in detail).

such activity has been associated to an important inhibitory roleplayed by laser on the synthesis of prostaglandin and TNF-a [19],and mast cell degranulation [20].

Polymorphonuclear neutrophils (PMN) are important players ininnate and acquired immunity. The MPO, a heme-containingenzyme involved in the generation of reactive oxygen speciesand oxidation of biomolecules [21], is stored in large amount inazurophilic granules of resting PMN cells [22]. Therefore, somestudies have used the quantification of tissue MPO as an inflamma-tory marker to estimate the accumulation of PMN [13].

We found that 17.5 J/cm2 LLLT did not promote significantchanges in the MPO content in the laryngeal tissues, suggestingthat this protocol of LLLT is not efficient in modulating themigration of PMN leukocytes into the injured area. Along withour findings, Ribeiro et al. [23] observed no significant differencein the histological content of PMN leukocytes in rats treated with16.0 J/cm2 in wound healing assay. These findings are suggestivethat low fluencies of LLLT are ineffective in controlling the acuteinflammatory response in cases when the phlogistic stimuli are in-tense and continuous.

However, the combined protocol 17.5/10.0 J/cm2 provided sub-stantial decrease in the influx of neutrophils in this model on the1st day of observation. In addition, histopathological analysis ofthe irradiated laryngeal tissues showed that combined 17.5/10.0 J/cm2 protocol, but not 17.5 J/cm2 protocol, induced signifi-cant decrease in the intensity of the inflammatory response andmore rapid transformation of acute into chronic inflammation, aswell as accelerated the development of the granulation tissue. Suchobservation might represent different patterns of laser wavelengthpenetration in the tissues. Ranges of 600–700 nm wavelengthshave been employed in treating superficial tissues, whereas 780and 950 nm wavelengths have been applied for deeper damagedtissues [24,25]. Thus, it is possible to speculate that the synergicassociation of the 17.5 J/cm2 at 780 nm and 10.0 J/cm2 at 660 nmmight have minimized the inflammatory content at both superfi-cial and deep esophagus tissues. In accordance, Santos et al. [25]

ctions of each animal, n = 6/group). (a) CTR – non-intubated rats. In detail, collagene in detail; (c) NLT17.5 – intubated rats treated with 17.5 J/cm2 laser irradiation.treated with 17.5/10.0 J/cm2 laser irradiation. Note exuberant granulation tissue

Fig. 4. Histological sections stained in toluidine blue, showing metachromatic mast cells in the laryngeal connective tissue of the experimental groups (three histologicalsections of each animal, n = 6/group). (a) CTR – non-intubated rats; (b) NGI – intubated rats; (c) NLT17.5 – intubated rats treated with 17.5 J/cm2 laser irradiation; and (d)NLT17.5/10.0 – intubated rats treated with 17.5/10.0 J/cm2 laser irradiation. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)

Fig. 5. The effect of LLLT on mast cells count after 7 days. Rats were separated ingroups: CTR – non-intubated rats; NGI – intubated rats; NLT17.5 – intubated ratstreated with 17.5 J/cm2 laser irradiation; and NLT17.5/10.0 – intubated rats treatedwith 17.5/10.0 J/cm2 laser irradiation. All values are expressed as mean ± SEM.Statistical analysis by ANOVA followed by Bonferroni’s test. �p < 0.001 vs. CTR group(n = 6/group).

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showed that the combination of different laser intensities is able inincrease the resolution of inflammation, on would model in rats.

Furthermore, since laser irradiation promotes strong stimula-tory effects on fibroblasts [26] and endothelial cells proliferation[27], it is possible to suggest that the granulation tissue formationis likely secondary to those modulatory properties. Besides, theearlier chronification of the inflammatory reaction might have re-sulted of the mitogen effects of LLLT on lymphocytes, particularlyon T cells, as previously reported [17]. Also, the lack of ulcerativeareas in NLT17.5/10.0 group suggests that the combined LLLTprotocol induced rapid epithelization of mucosal surface. Thesefindings are supported by previous studies demonstrating thatlow level laser in low fluencies was shown to be effective in stim-ulating the migration of keratinocyte along wound surfaces [18].Furthermore, LLLT was proved to act directly on keratinocyte-pro-moting epithelial cell proliferation in vitro [28].

Mast cells are thought to play a fundamental role on themodulation of the inflammatory response. These cells produce

and release a wide range of chemical mediators which are relatedto vascular changes (vasodilation and increase in the vascularpermeability) and chemotactic signalizing, in order to facilitatethe influx of leukocytes into damaged tissues, particularly in theacute phase of the inflammatory response [29]. It has been previ-ously reported a close relationship between the intensity of mastcell degranulation and the long-term persistence of the acuteinflammatory reaction [30].

We found that mast cells count was significantly decreased innon-irradiated and in 17.5 J/cm2-irradiated rats, but not in animalstreated with combined LLLT (17.5/10.0 J/cm2). It is well-estab-lished that toluidine blue stain interacts with cationic granules ofmast cells, and that degranulation process is supposed to avoidmast cells identification by this histochemical method [31].Therefore, it is possible to suggest that NSGI leads to local contin-uous mechanical stimulation and mast cell degranulation. Besides,the subacute inflammatory infiltrate seen in these groups appearsto support this hypothesis since mast cell degranulation extendschemotactic signalizing over the time course of the inflammatoryresponse and leads to long-term persistence of neutrophils-richinfiltrate [32].

It has been reported that LLLT seems to be able to up-regulatemast cells degranulation by promoting increase of intracytoplas-mic levels of mitochondria-derived Ca2+ [33,34]. Therefore,although laser irradiation at low fluences has also been demon-strated to increase mast cells counts in injured tissues, it wouldlikely be a result of laser-induced direct migration of mast cells[35]. Nevertheless, Pereira et al. [20] have recently reported thatsuch increase in mast cells number promotes amplification of theacute inflammatory response. On the contrary, we showed thatNLT17.5/10.0 group presented well-developed granulation tissueand lack of neutrophils infiltration. We hypothesize that combinedLLLT wavelength could have stimulated the acute phase of theinflammatory response, accelerating the biological events of thehealing process, and promoting earlier reconstitution of mast cellspopulation in the injured tissues.

Fig. 6. Histological sections stained in sirius red, analyzed under polarized light (three histological sections of each animal, n = 6/group), showing the pattern of collagendeposition in both esophageous (esg) and laryngeal (lrx) connective tissues in the experimental groups. (a) CTR – non-intubated rats; (b) NGI – intubated rats; (c) NLT17.5 –intubated rats treated with 17.5 J/cm2 laser irradiation; and (d) NLT17.5/10.0 – intubated rats treated with 17.5/10.0 J/cm2 laser irradiation. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

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The fact that the single protocol of 17.5 J/cm2 at 780 nm failedin providing similar biological effects supports the hypothesis ofthis wavelength promotes biomodulatory effects in deep esopha-geous tissues, but is ineffective to modulate the inflammatory re-sponse at laryngeal superficial ones. In fact, these results seem tobe in agreement with the pathophysiological mechanisms involvedin the development of reflux laryngitis in this experimental model.Thus, as long as the inflammatory response in the laryngeal tissuesis likely promoted by a secondary stimulus induced by gastro-esophagic reflux, the laser activity concentrated only in the deeperesophageous sites is not expected to interfere in the dynamics oflaryngeal mast cells degranulation.

Another hypothesis is that each wavelength is effective within anarrow therapeutic window, thus certain wavelengths may bemore effective than others for treat inflammatory processes insome diseases [7,36]. The red laser has been demonstrated to begenerally effective with lower doses than infrared laser whentreating mucosa, even though both wavelengths seem effective[37].

It is also possible to speculate that the significant difference be-tween the two laser groups might have resulted from either thedoubling of the total irradiation time in the combined therapy(NLT17.5/10.0 group). Therefore, the lacks of significance for infra-red laser in NLT17.5 group (MPO activity and mast cell degranula-tion) may be result of underpowered sample. We must alsoconsider that in the present study, the irradiation points were ap-plied within 0.5 cm of each other, allowing for considerable over-lap between the three irradiated areas.

The sirius red method, largely used for collagenization studies,is based on the presence of alkaline aminoacids in the collagenmolecules that strongly react with the acid stain (sirius red),increasing the birefringence of the normal aggregated collagenmolecules, when analyzed under polarized light. This stain hascontributed substantially for the identification and comprehensionof the collagen and its function, since it allows the differentiation

between the type I (intense yellow-orange and reddish birefrin-gence) and type III collagen (mild greenish birefringence) [38].Therefore, resultant pattern and disposition of collagen fibers ana-lyzed using polarized light has been employed to assess thedynamics of the connective tissue repair [39].

In this study, the substantial loss of collagen seen NGI andNLT17.5 groups might have resulted of the activation of collagen-degrading metalloproteinases, released by the high content of theinflammatory PMN neutrophils [40]. Besides, elevated levels of de-graded collagen appear to downregulate type I procollagen synthe-sis [41].

On the other hand, the dense network of both types I/III colla-gen fibers observed in NLT17.5/10.0 group suggests that combinedlaser protocol promoted stimulatory effect on the fibrogenesis,accelerating the collagenization dynamics. Laser-induced collage-nization improvement during wound healing has been previouslydescribed in other investigations [25], and it has been associatedto a direct activity of LLLT on the fibroblastic metabolism and col-lagen synthesis [42]. Moreover, both stimulatory and inhibitory ef-fects of laser arrays on fibroblastic activity have been previouslyreported, depending on the wavelength, dose, and tissue thickness[43].

In addition, the persistence of some type III collagen fibrils inthis very group might be related to the modulatory activity ofthe laser irradiation on the granulation tissue, since such collagenmolecules appears to be involved in the orientation of the prolifer-ation and migration of endothelial cells during the development ofthis step of the healing process [44].

Furthermore, it also suggests a slower replacement of the deli-cate type III fibrils for more resistant gross thicker type I collagen incomparison to the other groups. The relevance of this finding layson the fact that early replacement of type III collagen and excessiveproduction of type I collagen, as observed in NLT17.5 group, mightfurther lead to the formation of undesirable hypertrophic scars andkeloids [45].

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In conclusion, we demonstrated that the combined 17.5 J/cm2 at780 nm and 10.0 J/cm2 at 660 nm successfully modulated earlyinflammatory response in experimental model of nasogastric intu-bation-induced reflux laryngitis. The possible mechanism involvesthe reduction in neutrophil infiltration, modulation of mast cellsdegranulation, and improved granulation tissue formation and col-lagen deposition in the injured area. Nevertheless, further investi-gations are required in order to clarify the precise mechanismsunderlying the effects of low level laser therapy on the experimen-tal model of reflux laryngitis.

Acknowledgements

This study was supported by Coordenação de Aperfeiçoamentode Pessoal de Nível Superior (CAPES), Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq), and Fundação deApoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe(FAPITEC/SE). R.R. Marinho received grants from CAPES. R.M. Matosreceived grants from CNPq. J.S. Santos received grants from FAPI-TEC/SE. R.A. Ribeiro and S.M. Thomazzi are recipients of CNPq pro-ductivity grants.

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