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For Peer Review
Effect Anti-inflammatory of Low-Level Laser Therapy (660 nm) in the Early Phase in Carrageenan- Induced Pleurisy in Rat
Journal: Lasers in Surgery & Medicine
Manuscript ID: LSM-07-0274
Wiley - Manuscript type: Research Article
Date Submitted by the
Author: 17-Nov-2007
Complete List of Authors: Boschi, Emerson; PUCRS, Biofisica Leite, Carlos; PUCRS, Biofisica
Saciura, Vasyl; PUCRS, Biofisica; PUCRS, Biofísica Caberlon, Eduardo; PUCRS, Biofisica
Lunardelli, Adroaldo; PUCRS, Biofisica Bitencourt, Shanna; PUCRS, Biofisica Melo, Denizar; PUCRS, Biofisica Oliveira, Jarbas; PUCRS, Biofisica
Key Words: Inflammation, LLLT, Pleurisy
John Wiley & Sons, Inc.
For Peer Review
Original Article 11-17-2007
Effect Anti-inflammatory of Low-Level Laser Therapy (660 nm) in the
Early Phase in Carrageenan- Induced Pleurisy in Rat
Emerson S. Boschi (1), Carlos E. Leite (1), Vasyl C. Saciura (1), Eduardo
Caberlon, MS (1), Adroaldo Lunardelli, MS (1), Shanna Bitencourt, MS (1),
Denizar A.S. Melo, PhD (1), Jarbas R. Oliveira, PhD (1).
(1) Laboratório de Pesquisa em Biofísica Celular e Inflamação – Pontifícia
Universidade Católica do Rio Grande do Sul (PUCRS) Av. Ipiranga 6681
prédio 12C sala 263 CEP 90619-900 – Porto Alegre – RS, Brazil
Corresponding author:
Dr. Jarbas Rodrigues de Oliveira
Pontifícia Universidade Católica do Rio Grande do Sul
Laboratório de Pesquisa em Biofísica e Inflamação
Av. Ipiranga 6681 prédio 12C sala 263 CEP 90619-900
Porto Alegre – RS, Brazil
Phone: + 55 51 3320 3500 extension line 4147
E-mail: jarbas@pucrs.br Page 1 of 35
John Wiley & Sons, Inc.
Lasers in Surgery and Medicine
For Peer Review
Authors’ Address:Emerson Soldateli Boschi
Rua Santa Cecília, 1373/403 – Bloco A – POA. Phone: (051) 3332-0737 / 9965-3661
E-mail: esboschi@terra.com.br
Carlos Eduardo Leite
Rua Marco Pólo, 125/307 – POA. Phone: (051) 96443005
E-mail: cadufar@yahoo.com.br
Vasyl Custódio Saciura
Rua Aracy Froes, 257/412 – POA. Phone: (051) 3332-1795 / 91053739 E-mail: vasyl@terra.com.br
Eduardo Caberlon
Av. Protásio Alves, 2732/301. Phone: (051) 3334-1838 E-mail: ecaberlon@pucrs.br
Adroaldo Lunardelli
Av. Juca Bastista, 7346
Phone: (051) 3245-6011 / 9217-5566 E-mail: adroaldo@via-rs.net
Shanna Bitencourt
Rua Anita Garibaldi, 500/301 – POA. Phone: (051) 3330-6600 / 8121-7770 E-mail: shannab@ig.com.br
Denizar Alberto da Silva Melo
Rua Vasco da Gama, 1279/302 – POA. Phone: (051) 3320-3646 / 9258-5121 E-mail: dmelo@pucrs.br
Jarbas Rodrigues de Oliveira
Av Ipiranga, 6681 prédio 12C sala 263 – POA. Phone: (051) 3320-3500 extension line 4147 E-mail: jarbas@pucrs.br
Page 2 of 35
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ABSTRACTBackground and Objective: In the classic model of pleurisy there is little
evidence of anti-inflammatory effects from LLLT and dosage characteristic,
such as wavelength, total energy, number and pattern of treatment, with the
necessity for more studies. The purpose of this study was to investigate the
potential effect of low level laser therapy (LLLT) on modulating the
pro-inflammatory and anti-pro-inflammatory mediators of acute inflammation rat
pleurisy model.
Study Desing/Material and Methods: A sample of 48 female Wistar rats
were divided into control and experiential groups. An inflammation was
induced by carrageenan (0.2mL) injected into the pleural cavity. At 1, 2, and 3
h after induction the continuous wave (20 mW) diode laser of the InGaAlP
(660nm) type was used in laser groups with different doses and treatment
patterns, and with a total energy delivered of 0.9, 2.1 and 4.2 joules. At 4 h
after the exudate volume, total and differential leukocytes, protein
concentration, NO, IL-6, IL-10, TNF- and MCP-1 were measured from the
aspirated liquid.
Results: Any treatment pattern and quantity of energy can reduce a
significantly volume of the exudate (P<0,05). The final energy lower than 2.1
joules reduces significantly (P<0,05) only NO, IL-6, MCP-1 and IL-10, while
2.1 joules reduce significantly all variables independently of the treatment
pattern. The neutrophil migration has straight correlation with the
TNF-(r=0,551) and NO (r=0,549) concentration.
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Conclusions: LLLT-660nm induced an anti-inflammatory effect characterized
by inhibition of both total and differential leukocyte influx, exudation, total
protein, NO, IL-6, MCP-1, IL-10 and TNF- , but keeping a highly
dose-dependent outline. Under these conditions, laser with 2.1 joules was more
effective than 0,9 joule and 4,2 joules.
Key Words: Inflammation, LLLT, Pleurisy
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INTRODUCTIONInflammation is a protective process essential for the preservation of the
integrity of the organisms in the event of physical, chemical and infectious
damage [1]. Acute inflammation is characterized by the classical sings of pain,
heat, redness and swelling, involving a complex series of events including
vasodilatation, increased permeability, fluid exudation and migration of
leukocytes to the site of the inflammation [2].
Carrageenan-induced pleurisy is a well-characterized experimental model of
inflammation that permits the quantification and correlation of both exudate
and cellular migration with changes of other inflammatory parameters [3]. The
major characteristic of this model in the rat is the biphasic profile of this
inflammatory reaction, where early (4 h) and late (48 h) phases of both cell
migration and exudation are clearly observed [4]. Thus, this model constitutes
a biologic system suitable for the investigation of possible correlation
occurring between cell migration, fluid leakage, nitric oxide (NO), chemokine,
pro-inflammatory and anti-inflammatory cytokines.
One of the early cellular events in inflammation is the margination of
leukocytes, primarily neutrophils. In addition, NO plays an important role in
inflammation such as plasma exudation and leukocyte infiltration. The NO
synthase (NOS) inhibitors can reverse several classic inflammatory symptoms
[5].
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The maintenance of leukocyte recruitment during inflammation requires
intercellular communication between infiltrating leukocytes and the
endothelium. These events are mediated by the generation of early response
cytokines, e.g., interleukin (IL)-1 and tumor necrosis factor (TNF- ), the
expression of cell-surface adhesion molecules and the production of
chemotactic molecules, such as chemokines [6].
Chemokines are a family of structurally related glycoproteins with potent
leukocyte activation and/or chemotactic activity. Most chemokines are
produced in response to a variety of inflammatory stimuli, including the
early-response cytokines, TNF- , IL-1, C5a, leukotriene B4 and interferons [7].
Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that has
demonstrated to attract monocytes and neutrophils both in vitro and in vivo
and appears to be a key part of leukocyte emigration, serving as a promoter
of the transition from leukocyte rolling to adhesion on the endothelial surface
[8].
Representative inflammatory cytokines include TNF- , IL-1, IL-6, and IL-8.
The TNF- and IL-1 have been revealed to have many overlapping biological
activities in the inflammatory reaction: both cause fever, accumulation of
neutrophils in local tissues, induction of vascular adhesion molecule and
stimulation of acute phase protein synthesis [9]. Some of these responses by
TNF- and IL-1 are now known to be mediated by other inflammatory
mediators or secondarily induced cytokines such as IL-6 [10].
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The IL-6 is widely produced by several cells such as fibroblasts, endothelial
cells, keratinocytes, monocytes, T cells, mast and tumour cell lines, and cells
of neural origin. Thus, IL-6 is often used as a marker for systemic activation of
pro-inflammatory cytokines [11]. However, evidence also indicates that, under
certain conditions, IL-6 has both pro-inflammatory and anti-inflammatory
properties [12]. Apart from this, there is also experimental evidence indicating
that IL-6 down-regulates the synthesis of IL-1 and TNF- [13].
The IL-10 is known as the most important anti-inflammatory cytokine found
within the human immune response. Previous data has shown that IL-10 is
capable of blocking the inflammatory response induced by several
inflammatory stimuli in different models [11].
The conventionally used therapies for inflammation; non-steroidal
anti-inflammatory drugs (NSAID’s), have very important role in managing pain and
acute inflammatory conditions [14], though with rather discouraging profile of
side effects [13]. Even the anti-inflammatory drugs, cyclo-oxygenase 2
(COX-2) inhibitors, are not devoid of adverse effects [15].
Despite this, alternative physical techniques such as low-level laser therapy
(LLLT) has been used clinically, among other indications, for its proposed
anti-inflammatory effects, pain relief and acceleration of the regeneration of
damaged tissues [16]. Although in the past decade several studies have
examined the effects of LLLT, the treatment protocols used included
enormous variations in parameters (such as wavelengths, energy and power
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densities, wave modes, number of treatments), which make it difficult to
assess the optimum treatment parameters in each case [17].
However, more recent studies have been able to find some dose-dependent
effects on TNF- [18] and prostaglandin E2 level [19], besides reduction of the
oedema [20,21], neutrophils migration [21], nitric oxide synthase (iNOS)
expression [22] and COX-2 mRNA expression [23] in the different
inflammatory experimental models after LLLT. The efficacy of LLLT radiation
as an anti-inflammatory therapy is controversial. The interaction of radiation
with the biological system occurs at the cellular level, but the mechanisms
involved are yet unknown.
Therefore, the present study was designed to explore the potential effects of
LLLT applied at different points with dose variation on the modulation of the
pro-inflammatory and anti-inflammatory mediators of acute inflammation in
carrageenan-induced pleurisy in rat model.
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MATERIALS AND METHODSAnimals
Adult female Wistar rats (Rattus norvegicus) bred in our laboratory (around
3-4 months old, weighing 180g-220g) were used, all of the same ancestry and
socialization with free access to food and water. Animals were divided into six
groups composed of eight rats each. The rats were maintained in accordance
with the “Guiding Principles in the Care and Use of Animals” approved by the
Council of the American Physiological Society.
Carrageenan-induced pleurisy
Rats were anesthetized with a mix of Ketamina (80mg/Kg) and Xilazina
(12mg/Kg). Saline 0.2 mL (saline group) or saline containing 2%
-carrageenan (Cg) 0.2mL (-carrageenan group and Cg + laser groups) was
injected into the pleural cavity at the level of the sixth left intercostal space. At
4 h after the intrathoracic injection, the animals were sacrificed with
decaptation and the pleural cavity was opened. The liquid that had
accumulated in the pleural cavity was washed with 2.0 mL of sterile saline
solution (NaCl 0.9%) containing 1% EDTA in the aspirated liquid. All exudate
contaminated with red blood cells was discarded [2].
Exudate Analysis
The volume of the exudates was measured and the result expressed by
subtracting the volume injected into the pleural cavity (2.0 mL of solution) from
the total volume aspirated. Total leukocytes were diluted in Thoma solution
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(1:20) and counted in a Neubauer chamber using light microscopy.
Cytological slide smears stained with May-Grünwald/Giemsa were used for
differential leukocyte counts in a light microscope [24]. The pleural liquid
removed from the rats was centrifuged at 1200 x g for 10 min and the protein
concentration measured by the Biuret technique. NO is a very unstable
radical, rapidly metabolized from nitrate to nitrite in the presence of oxygen
[25]. Therefore, the amount of NO in the exudate was analyzed using the
Griess reaction that measured nitrite, as previously described [26]. The frozen
cell-free supernatant of the pleural fluid was thawed, and IL-6, IL-10,
TNF-and MCP-1 were determined using a rat enzyme-linked immunosorbent assay
(ELISA) kit (Biosource, Camarillo, CA).
Laser Irradiation
The continuous wave diode laser of InGaAlP type (model
Endophoton-LLT-010 - KLD Biosistemas Equipamentos Eletrônicos Ltda) with an output power
of 20mW and wavelength of 660 nm (visible red) was used. The spot size was
0.035 cm2 and the power density was 0.571 W/cm2. Table 1 shows the laser
parameters used in our experiment. Different doses were used for each of the
four groups: group with 1 point (Cg + 1 point laser with 21 J/cm2), group with 3
points (Cg + 3 points laser with 3 J/cm2), group with 7 points (Cg + 7 points
laser with 3 J/cm2) and group with 14 points (Cg + 14 points laser with 3
J/cm2). All the experimental groups were irradiated with LLLT from a spot in
contact with the skin at 1, 2 and 3 h after pleurisy induction. Thus, the total
energy delivered from the three treatment sessions was 2.1, 0.9, 2.1 and 4.2
joules, respectively. The animals received the irradiation at the left thoracic
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wall with different treatment methods according to the group: group of 1 point
was irradiated with LLLT where the pleurisy was induced; group of 3 points
received LLLT around the induction area; group of 7 points was irradiated, in
which 1 point was on the induction local and 6 points were symmetrically
distributed around the area; in group of 14 points they were distributed in a
craniocaudal direction of crescent 2, 3, 4 and 5 points involving all thoracic
wall.
Statistical Analysis
The results were evaluated statistically by analysis of variance (ANOVA) with
LSD test post hoc using SPSS (Statistical Package for the Social Sciences)
12.0 software and were expressed as the means ± standard error of means
(SEM). The correlation between the variables and the neutrophil infiltration
into the pleural cavity was analyzed by using Pearson Correlation Linear. The
level of statistical significance was defined as P < 0.05.
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RESULTSAccording to the analysis of this study, the rats injected with carrageenan in
the pleural space developed an acute pleurisity after 4 h with a significant
increase in all variables when compared to the saline group. This episode
shows that carrageenan causes a potential inflammatory reaction.
Figure 1 shows the protective effect of LLLT on the inflammatory parameters,
such as: protein concentration, exudate volume, the amount of leukocytes and
neutrophils in the pleural liquid of the saline group, carrageenan and in the
four groups irradiated with different laser doses and treatment methods.
Protein
Protein concentration (fig 1A) showed a significant decrease (P<0.05) in laser
groups with 1 point (1.01 ± 0.11), 7 points (0.94 ± 0.03) and 14 points (1.13 ±
0.10) when compared to the carrageenan group (1.42 ± 0.04). It was
observed that laser group with 3 points (1.47 ± 0.07) did not modified the
concentration of proteins that had migrated to the pleural cavity.
Exudate volume
In relation to the exudate volume (fig 1B) collected 4 h after the pleurisy
induction, it was observed a significant reduction in the four groups irradiated
with LLLT when compared to the carrageenan group (1 point: 0.32 ± 0.03; 3
points: 0.68 ± 0.07; 7 points: 0.60 ± 0.08 and 14 points: 0.52 ± 0.06 vs Cg:
0.98 ± 0.04, P<0.001). Besides that, laser group with 1 point (2,1 joules) was
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more effective than laser group with 7 points (2,1 joules) in reducing pleural
exudate volume (P=0.002).
Total leukocytes
All groups irradiated with LLLT, except the group with 3 points, showed a
significant decrease in the total number of leukocytes when compared to the
carrageenan group (1 point: 35.03 ± 4.07; 3 points: 63.25 ± 5.69; 7 points:
37.21 ± 2.35 and 14 points: 46.22 ± 3.81 vs Cg: 70.67 ± 3.90, P=0.001), (fig
1C). Based on this, the group that received a final dose of 0.9 joule (group 3
points) did not demonstrate reduction in the leukocyte cells infiltration to the
inflammatory site of the lesion (P=0.176).
Neutrophils
The reduction in the inflammatory acute phase response, represented by a
lower migration of polymorphonuclear-neutrophil cells (PMNs), showed a
mean of 50% reduction of these cells in the groups 1, 7 and 14 points that
received higher or equal to 2.1 joules energy when compared to the
carrageenan group or the laser group with 3 points (1 point: 31.09 ± 3.60; 7
points: 31.61 ± 1.90 and 14 points: 36.63 ± 4.20 vs Cg: 61.11 ± 1.76,
P<0.001). The group 3 points did not demonstrate significant reduction in the
neutrophil infiltration (58.30 ± 5.60, P=0.568), (fig 1 D).
NO
The concentration of nitric oxide (NO) in the pleural cavity (fig 2A) presented a
significant reduction in the groups irradiated with laser when compared to the
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carrageenan group (1 point: 49.40 ± 2.53; 3 points: 55.25 ± 2.49; 7 points:
49.44 ± 2.22 and 14 points: 55.70 ± 2.88 vs Cg: 86.02 ± 3.86, P=0.001). It is
noticed that in laser groups with 1 and 7 points the reduction approximates
the basal level values of the saline group (49.78 ± 5.63).
IL-6
In relation to the pro-inflammatory cytokine, it was also observed a reduction
in the concentration of IL-6 in the irradiated groups after the application of
energy when compared to the one that received only carrageenan (1 point:
7298.82 ± 178.99, 3 points: 8596,92 ± 231.76, 7 points: 7955,80 ± 478.74 and
14 points: 14587.74 ± 2613.69 vs Cg: 25357.77 ± 5451.93, P<0.001). This
important mediator presented sensibility to LLLT irradiation independently of
the chosen treatment method and energy dose (fig 2B).
MCP-1
The chemokine MCP-1 had a significant decrease in laser groups with 1 point
(1131.25 ± 175.74, P=0.019), 3 points (1228 ± 209.39, P=0.025) and 7 points
(1434.50 ± 171.89, P=0.046) when compared to the carrageenan group
(3007.25 ± 1067.61), but in laser group with 14 points (2578.85 ± 741.09,
P=0.591), where the total final energy was equal to 4.2 joules, a significant
reduction was not verified in the measured values from the pleural liquid count
(fig 2C).
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IL-10It was verified a significant reduction of IL-10 concentration in the irradiated
groups with 1 point (560.05 ± 70.18, P=0.002), 3 points (690.76 ± 114.44,
P=0.007) and 7 points (855.87 ± 239.78 P=0.049) when compared to the
carrageenan group (1436.15 ± 305.68). In the group with 14 points (957.42 ±
216.25), IL-10 did not suffer any modification on the measured values
showing that a more local treatment pattern tends to intervene in the final
result (P=0.088), (fig 3A).
TNF
The concentration of TNF- (fig 3B) decreased significantly in the LLLT
irradiated groups with 1 point (523.90 ± 122.21), 7 points (298.05 ± 69.21) and
14 points (509.05 ± 146.07) when compared to the carrageenan group
(1535.20 ± 402.81, P<0.001). The laser group with 3 points did not present a
decrease in TNF- levels when compared to the carrageenan group (1206.27
± 359.99 vs 1535.20 ± 402.81,P=0.336).
Correlation between neutrophil cell migration with the study variables
At 4 h after carrageenan-induced pleurisy it was observed a significant
increase in neutrophil migration to the pleural cavity in the carrageenan group
when compared to the saline group (fig 1D). With regard to all variables
correlated to the neutrophil infiltration in the pleural cavity, only MCP-1
(P=0.060) and IL-10 (P=0.092) did not present a significant statistics-level. In
our experimental model, the first inflammatory mediator to show a main
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correlation with neutrophil infiltration was TNF- (r=0.551) and the second
was NO (r=0.549). The results are summarized in Table 2.
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DISCUSSIONImmediately after an acute injury event the body, in response to the disruption
of vascular integrity, soft tissue, connective tissue and neurological
processes, initiates a series of biological responses. The inflammatory
reaction consists of both vascular and cellular events. Injury responsive
components such as mast cells, bradykinins and prostaglandins are activated
along with the vascular responses and cellular membrane reactions. All of
these combined processes and events are represented by the symptoms of
edema, inflammation, pain and functional debility [4,11,27].
The carrageenan is a polysaccharide frequently used to induce acute
inflammatory reaction in animal experimental models. Winter (1962)
introduced the use of carrageeenan as an irritant agent of rat paw edema,
becoming the first and most popular method to evaluate new
anti-inflammatory therapies with a hydroplethysmometer for inflammated paw
volume measurement [28].
The paw edema induced by carrageenan is an useful method to evaluate
acute inflammation, since edema peak occurs within 3 to 5 hours [29].
However, this technique has some limitations on measuring inflammatory
cells, proteins and chemical mediators, once it is not possible to extract the
inflammatory exudate.
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Despite this, carrageenan-induced pleurisy in rats permits quantifying the
volume and protein concentration of the exudate formed, besides the
evaluation of the inflammatory cell migration to the pleural cavity [30]. This
kind of pleurisy is used to investigate acute inflammation pathophysiology and
also to evaluate anti-inflammatory therapies efficacy.
The inflammatory response that occurs after carrageenan injection into the
pleural cavity is characterized by cellular infiltration, mainly composed of
neutrophils (approx. 90%) and to a lesser extent of monocyte/macrophages
[27].
Phototherapy with LLLT has achieved high success in recent years. It has
gained the approval of the U.S. Food and Drug Administration (FDA) and is
gradually earning its place in mainstream medical practice [33].
Many studies have suggested advantages of the biomodulatory effects of
LLLT on the inflammatory process, wound healing and pain relief, but the
basic mechanism of these effects remains unclear. LLLT has been shown to
reduce the duration of acute inflammation and pain, just as to accelerate
tissue repair in tendon and muscle injuries [32].
Laser therapy uses different wavelengths of the visible and near-infrared
spectra incluind HeNe (632.8nm), GaAlAs (780-870 nm), InGaAlP (630-685
nm) and GaAs (904 nm) [34,35]. Many researchers have attempted to
understand the action of LLLT, as well as to determine the most appropriate
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wavelength, period of irradiation, number of treatments, energy density and
energy total.
Albertini et al. (2004) have demonstrated that LLLT with diode Ga-Al-As (650
nm) employed with a continuous power of 2.5 mW and energy density of 2.5
J/cm² can reduce edema caused by carrageenan-inflammation in rat paw. In
the anti-inflammatory response it has shown similar performance to the
sodium diclophenac when intraperitoneally administrated with a dose of 1
mg/kg. In this study the authors verified that LLLT did not inhibit the
carrageenan-induced edema in adrenalectomized rat models, suggesting that
the mechanism of action can be related with the
hypothalamo-hypophyseal-adrenal endocrine system and, as a consequence, with the release of
endogenous corticoids [20].
Recently, Martins et al. (2005) described an inibitory effect of LLLT (650 nm –
2.5 mW) on leukocyte (neutrophil) migration to the pleural space in the
carrageenan-induced pleurisy in mice with energy density of 1 J/cm2, 2,5
J/cm2 and 5 J/cm2, applied on the 1st, 2nd and 3rd hour after the inflammatory
induction. It was observed that the total dose of 7.5 J/cm² (0.6 joules) was
more effective [21].
Based on these studies that have evaluated the anti-inflammatory action of
LLLT, the dosage of 2.5 J/cm² with power of 2.5 mW has demonstrated to
reduce edema and inflammated cells when applied local radiation on the
inflammated site at 1, 2 and 3 h after the carrageenan induction, knowing to
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