light-emitting diode therapy induces analgesia and decreases spinal cord and sciatic nerve tumour...

ORIGINAL ARTICLE

Light-emitting diode therapy induces analgesia and decreasesspinal cord and sciatic nerve tumour necrosis factor-a levelsafter sciatic nerve crush in miceF.J. Cidral-Filho1,2, D.F. Martins1,2, A.O.O. Moré1,2, L. Mazzardo-Martins1,2, M.D. Silva1,2, E. Cargnin-Ferreira3,A.R.S. Santos1,2

1 Laboratório de Neurobiologia da Dor e Inflamação, Centro de Ciências Biológicas, Departamento de Ciências Fisiológicas, Universidade Federal de

Santa Catarina, Florianópolis, Brazil

2 Programa de Pós-Graduação em Neurociências, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil

3 Laboratório de Marcadores Histológicos, Instituto Federal de Educação Ciência e Tecnologia de Santa Catarina – Campus Lages, Lages, Brazil

CorrespondenceAdair R. S. Santos

E-mail: [email protected],

[email protected]

Funding sourcesThis study was supported by grants from

Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq), Coordenação

de Aperfeiçoamento de Pessoal de Nível

Superior (CAPES) and Fundação de Amparo à

Pesquisa e Inovação do Estado de Santa

Catarina (FAPESC), Brazil.

Conflicts of interestNone declared.

Accepted for publication17 December 2012

doi:10.1002/j.1532-2149.2012.00280.x

Abstract

Background: Neuropathic pain is severely debilitating and resistant topharmacological approaches; therefore, the study of therapies tocomplement its treatment is especially relevant. In a case report study,light-emitting diode therapy (LEDT) has shown analgesic activity as well asreduced the expression of pro-inflammatory cytokines in a rabbitosteoarthritis model and in calcaneal tendinitis in rats. Although LEDTstimulated morphofunctional recovery after nerve injury in rats, its effectagainst neuropathic pain has not been tested.Methods: To that purpose, mice under anaesthesia were subjected to thesciatic nerve crush (SNC) model. On the seventh post-operative day, afterdetermining analgesic dose (energy density in joules), LEDT (950 nm,80 mW/cm2, 2.5 J/cm2) was irradiated, daily for a period of 15 days, on theskin over the crush site.Results: Compared with the SNC group, LEDT reduced mechanicalhypersensitivity but not cold hypersensitivity which is induced by SNC,decreased spinal cord and sciatic nerve levels of tumour necrosis factoralpha (TNF-a) but did not alter interleukin (IL)-1b and IL-10 levels, andfinally, failed to accelerate motor functional recovery and morphologicalnerve regeneration.Conclusion: Taken together, these data provide first-hand evidence ofLEDT effectiveness against neuropathic pain induced by SNC, withcorresponding decrease of pro-inflammatory cytokine levels, both in thesciatic nerve and in the spinal cord, although at a small analgesic dose,LEDT failed to accelerate nerve regeneration.

1. Introduction

Neuropathic pain (NP) is defined as pain arising as adirect consequence of peripheral or central nerveinjury due to acute events or systemic diseases (Gabayet al., 2011; Zhuo et al., 2011). With a prevalence of upto 3% of the world population (Dworkin et al., 2003)and serious impact upon quality of life (Poliakov andToth, 2011), NP is severely debilitating (Kim and

Moalem-Taylor, 2011) and is resistant to pharmaco-logical approaches (Baptista et al., 2007; Attal et al.,2010), often producing undesirable side effects or inad-equate analgesia (Niederberger et al., 2008; Anandet al., 2011).

NP depends on an up-regulation of a complexnetwork of molecules in the spinal cord and dorsal rootganglia (Lee et al., 2010), including pro-inflammatorycytokines [e.g., tumour necrosis factor-a (TNF-a) and

1193Eur J Pain 17 (2013) 1193–1204 © 2013 European Federation of International Association for the Study of Pain Chapters

interleukin-1b (IL-1b)] (Watkins et al., 2001; Milliganet al., 2003). The model of sciatic nerve crush (SNC)not only produces intense hyperalgesia that mimics NPbut is also suitable for the study of nerve regeneration(Varejao et al., 2004).

Light-emitting diode therapy (LEDT) is a non-invasive form of phototherapy widely used as a thera-peutic tool in tissue healing and soft tissue injuries(Vinck et al., 2005). Other forms of phototherapyinclude low level laser (LLL) and polarized lighttherapy (PLT).

Although all types of phototherapy are regularlyused in clinical settings, only a few studies demon-strate their analgesic effect. LLL, for instance, pro-duced analgesia in experimental models of NP in rats(Giuliani et al., 2004; Lorenzini et al., 2010; Bertoliniet al., 2011; Hsieh et al., 2012). Additionally, LLL waseffective in the treatment of carpal tunnel syndromeparaesthesia, improved power of hand grip and theelectrophysiological parameters in a clinical study(Shooshtari et al., 2008).

PLT induced analgesia in acute nociception modelsin mice (Tamarova et al., 2005, 2009; Limansky et al.,2006), and in a controlled clinical study, it effectivelytreated various chronic pains in conjunction withnerve or local block (Huang et al., 2012).

Conversely, LEDT analgesic effect has not beeninvestigated in pre-clinical models of pain. Neverthe-less, LEDT presented anti-inflammatory activity andhas reduced the expression of TNF-a in a rabbitosteoarthritis model (Oshima et al., 2011), decreasedthe levels of TNF-a and IL-1b mRNA at the site ofcalcaneal tendinitis in rats (Xavier et al., 2010a);inhibited the expression of cyclooxygenase-2 andprostaglandin E2 and pro-inflammatory cytokines in

vitro (Lim et al., 2007; Choi et al., 2012); as well asstimulated morphofunctional recovery after SNC inrats (Serafim et al., 2012). Moreover, Costa et al.(2008) demonstrated that LEDT when applied to acu-puncture points was effective – at varying degrees – inthe treatment of 15 patient with chronic pain.

In light of these data, this study was designed toanswer the following questions: (1) is LEDT effectiveagainst NP induced by SNC in mice? (2) Is this effectdose-dependent? (3) Does a small analgesic dosemodulate pro- and anti-inflammatory cytokines levelsin the sciatic nerve and spinal cord of SNC mice? (5)Finally, does it accelerate morphological and motorfunctional neuronal regeneration?

2. Methods

2.1 Animals

Experiments were conducted using adult male Swissmice weighing 25–35 g, housed at 22 °C under a 12-hlight/12-h dark cycle (lights on at 06:00 h), withaccess to food and water ad libitum. The animals wereacclimatized to the laboratory for at least 1 h beforetesting. The experiments were performed after theapproval of the protocol (PP00437) by the Ethics Com-mittee of the Federal University of Santa Catarina andwere carried out in accordance with the current guide-lines for the care of laboratory animals and the ethicalguidelines for investigations of experimental pain inconscious animals (Zimmermann, 1983).

The number of animals and intensities of noxiousstimuli used were the minimum necessary to demon-strate consistent effects of treatments. Taking that intoconsideration, the animals used for the evaluation ofmechanical hypersensitivity were also used for thedetermination of the sciatic functional index (SFI) andsciatic static index (SSI), lower hind leg skeletalmuscle weighing as well as for histological and mor-phological analysis. Time course analyses of chronicLEDT effect upon mechanical hypersensitivity, coldhypersensitivity and determination of cytokineslevel were conducted, each with different animalgroups. All behavioural, histological and biochemicalanalyses were performed blinded with respect togroup assignment.

2.2 LEDT

Three experimental groups were used throughout theexperiments (n = 8): SNC, Sham and SNC + LEDT.During LEDT, the animals were contained in a plastictube with the tail and hind limbs protruding. During

What’s already known about this topic?• In a case report study, light-emitting diode

therapy (LEDT) has shown analgesic activity;• Reduced the expression of pro-inflammatory

cytokines in models of inflammation; and• Stimulated morphofunctional recovery after

nerve injury (dose of 4 J/cm2).

What does this study add?• It demonstrates that LEDT induced analgesia in a

model of chronic neuropathic pain and• Decreased TNF-a levels (sciatic nerve and spinal

cord);• Although analgesic dose (2.5 J/cm2) failed to

accelerate nerve regeneration.

Light-emitting diode therapy analgesic effect F.J. Cidral-Filho et al.

1194 Eur J Pain 17 (2013) 1193–1204 © 2013 European Federation of International Association for the Study of Pain Chapters

treatment, LED probe lightly touched the irradiationarea (1 cm2) located on the skin directly above theSNC site. LED device used in the experiments was aMOLIMEDpen© (MDT Bioelectronics, Bettwiesen,Switzerland) with 950 nm wavelength, irradiance of80 mW/cm2 and selected dose of 2.5 J/cm2 (32-s irra-diation). As the irradiation area is 1 cm2, the total doseapplied was 2.5 J.

In order to determine minimal analgesic dose(energy density in joules), additional animal groupswere used in the first experiment (Fig. 1): 1 J/cm2

(13-s irradiation) and 4 J/cm2 (50-s irradiation). Addi-tionally, to investigate if stress induced by restrainwould be responsible for LEDT analgesic effect, agroup of animals (n = 8) was treated with the LEDdevice turned off (animals were restrained for 32 swith the LED probe slightly touching the irradiationsite). SNC + LEDT group was treated daily from day 7to day 21 post-sciatic nerve crush.

2.3 Sciatic nerve crush

Surgical procedures were performed under deep ana-esthesia induced by a premixed solution of ketamine(100 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.). Aftershaving and asepsis of the skin with 10% povidoneiodine, the right sciatic nerve was exposed through askin incision extending from the greater trochanter tothe mid thigh distally. The sciatic nerve was then

crushed once for 30 s with a 2-mm-wide forceps, asdescribed in a previous report, with minor modifica-tions (smooth forceps) (Baptista et al., 2007). Thecrushing site was 1 cm above the nerve trifurcation toensure good reproducibility of the axonotmesis lesion.Sham group animals were subjected to the same sur-gical procedures, although the sciatic nerve was notcrushed.

2.4 Mechanical hypersensitivity

Mechanical hypersensitivity was assessed with vonFrey filaments (VFH; Stoelting, Chicago, IL, USA) withbending forces from 0.02 to 4 g. Fifty per centmechanical withdrawal threshold (the force in gramsto which an animal reacts in 50% of the presenta-tions) was determined according to the Dixon up-and-down method (Dixon, 1980; Chaplan et al., 1994)modified for mice (Sommer and Schafers, 1998). Oneach testing day, the mice were habituated in indi-vidual Plexiglas boxes (9 cm ¥ 7 cm ¥ 11 cm) on anelevated wire mesh platform for 1 h. Testing was ini-tiated with the 0.4-g filament. The filaments wereapplied and held for a period of approximately 3 sfrom underneath the grid floor perpendicular to theplantar surface. A positive response was recorded ifthe paw was withdrawn, in which case the nextweaker filament was applied and the next measure-ment was recorded. In the absence of a response, the

Figure 1 Effect of light-emitting diode therapy

(LEDT) on hypersensitivity induced by the

sciatic nerve crush (SNC) in mice. (A) Time

course of the effect of LEDT (950 nm, 1, 2.5

and 4 J/cm2) on mechanical hypersensitivity. (B)

Effect of chronic LEDT (950 nm, 2.5 J/cm2) on

mechanical hypersensitivity. Effect of chronic

LEDT (950 nm, 2.5 J/cm2) on cold hypersensitiv-

ity (C) on latency to reaction and (D) time of

reaction. ‘B’ corresponds to evaluation before

surgical procedure; ‘P’ indicates evaluation

before each treatment. Each group represents

the mean of eight animals, and the vertical

lines indicate the standard error of the mean.

***p < 0.001 when comparing SNC + LEDT with

the SNC group; ###p < 0.001 when comparing

SNC versus sham groups. Statistics: (A) at 0.5 h

– one-way analysis of variance (ANOVA) fol-

lowed by Newman–Keuls multiple comparison

test; (B–D) one-way ANOVA repeated mea-

sures followed by Newman–Keuls multiple

comparison test.

F.J. Cidral-Filho et al. Light-emitting diode therapy analgesic effect


next stronger filament was presented. This procedurecontinued until six responses in the immediate vicin-ity of the 50% threshold were obtained. The resultingsequence of positive and negative responses was usedto interpolate the 50% withdrawal threshold. Groupswere evaluated before the surgical procedure and onpost-operative days 7, 10, 13, 16, 19 and 21, 30 minafter LEDT.

In order to determine whether the analgesic effect ofLEDT is extended after repeated applications, inanother experiment the time course of the effect ofLEDT (950 nm, 1, 2.5 and 4 J/cm2) on mechanicalhypersensitivity on days 10, 13, 16 and 19 after SNCwas evaluated.

2.5 Cold hypersensitivity

Hypersensitivity to cold stimulus was assessed usingthe cold plate test (Cold/Hot Plate Analgesia Meter;AVS Projetos, São Paulo, Brazil), as designed byBennett and Xie (1988) with minor modifications. Theanimals were placed on a cold stainless plate in a spacesurrounded by clear Plexiglas (12 ¥ 20 ¥ 10 cm). Thetemperature of the cold plate was continuously moni-tored and kept constant (10 � 1 °C). The latency tothe first paw lifting, licking or shaking (pain-relatedbehaviours) of the right hind paw was recorded. Thecut-off time of the latency was set at 120 s to preventtissue damage. Groups were evaluated before surgicalprocedure and on post-operative days 7, 10, 13, 16, 19and 21, 30 min after LEDT.

2.6 Determination of cytokine levels

On the 13th day post-surgery (after seven consecutiveLED treatments), the lumbar spinal cord (L4–L5) andright sciatic nerve (distal portion) of the mice werecollected and used to estimate the cytokine levels byenzyme-linked immunosorbent assay (ELISA), withsample values corrected by protein levels (Mizgerdet al., 2001). Sample aliquots of 100 mL were used tomeasure TNF-a, IL-1b and IL-10 levels using mousecytokine ELISA kits from R&D Systems (Minneapolis,MN, USA), according to the manufacturer’s instruc-tions (TNF-a-DY410 kit, protein range of 31.25–2000 pg; IL-1b-DY401 kit, protein range of 15.62–1000 pg and IL-10-DY417 kit, protein range of31.25–2000 pg). The absorbance for the aforemen-tioned cytokines was measured using a microplatereader at 450 and 550 nm.

2.7 SFI and SSI

To investigate the possible effects of LEDT on motorfunction recovery, two footprint parameters were

obtained: toe spread (TS), the distance between thefirst and fifth toes, and print length (PL), the distancebetween the tip of the third toe and the most posteriorpart of the foot in contact with the ground.

Briefly, the animals were individually disposed on aglass corridor (48 ¥ 4.5 cm) with a mirror placedunderneath the apparatus at an angle of 45°. Thewalking pattern was recorded with a video camera(Panasonic Camcorder Digital PV-GS19; Panasonic,Newark, NJ, USA) while the mice walked through theglass runway. To analyse the footprints, 10 singleframes were used, 5 of each foot. This image wasloaded into a computer using a frame grabber asdescribed previously (Dijkstra et al., 2000; Baptistaet al., 2007). All evaluations of motor functionalrecovery were made via digital images, which werecaptured and analysed using the image analysis soft-ware Image-Pro Plus software 6.0 (Media Cybernetics,Bethesda, MD, USA).

The factors for each parameter (TS and PL) werecalculated with the formula: injured – uninjured/uninjured values. SFI was calculated as previouslydescribed by Inserra et al. (1998) according to the fol-lowing equation:

SFI = 118.9 ¥ TSF - 51.2 ¥ PLF – 7.5

Static footprints were obtained with the animals ona resting position. The TS and PL were estimated andgenerated the TS factor (TSF) and PL factor (PLF)(Baptista et al., 2007) which were used in the formula:SSI = 101.3 ¥ TSF - 54.03 ¥ PLF - 9.5.

In the SSI and SFI, a value of 0 corresponds tonormal function (i.e., both sides with the same func-tional status) and a value of -100 corresponds to com-plete loss of function of one side. Testing wasperformed before surgical procedure and on post-operative days 7, 10, 13, 16, 19 and 21.

2.8 Lower hind leg skeletal muscle mass

To investigate the possible effects of LEDT on lowerhind leg skeletal muscle mass (muscles innervated bythe tibial nerve, a ramification of the sciatic nerve,indirectly affected by the SNC injury), on the 21st daypost-injury, the mice were euthanized by cervical dis-location. The animals were then weighed and the rightsoleus and gastrocnemius muscle were excised andweighed. In order to correct for individual differences,data were calculated with the formula: Weight =muscle weight ¥ 10/animal whole weight, accordingto Vescovo et al.’s (1998) relative muscle weight defi-nition with minor adaptation.



2.9 Histology and morphological analysis

On the 21st day post-injury, after functional assess-ment, the mice were euthanized by cervical disloca-tion. The distal portion of the right sciatic nerve wasexcised and immediately immersed in a buffered fixa-tive solution of zinc-formalin (1.6% zinc chloride,4% formaldehyde, 20% calcium acetate) for 24 h.After fixation, the samples were placed in 5% potas-sium dichromate for 5 days and then put in runningtap water overnight to wash out the dichromatebefore dehydrating in graded concentrations ofethanol. All samples were embedded in paraffin wax.Sections of 5-mm thick were obtained, and the slideswere stained with Mason and ‘Oil Red’ (Bobinskiet al., 2011; Martins et al., 2011) as describedbelow.

All morphological analysis was performed blindedwith respect to group assignment. Once stained, theslices were observed and photographed under lightmicroscopy. Four parameters were quantified: (1)area of connective tissue (%); (2) area of myelinatedfibres (%); (3) density of myelinated fibres/area analysed; and (4) myelin sheath area (mm),were measured in photographs at 400 and1000 ¥ magnification. For myelin sheath thickness, arepresentative area where 10 whole myelinatedaxons could be counted was chosen (SupportingInformation Fig. S1a–d). The histological examina-tion was restricted to the endoneuro and the myelinsheath area. Fields with folds or poorly preservedtissue components in histological sections wereexcluded. Digital images were acquired using a lightmicroscope (Olympus, BX-41; Olympus, Melville,NY, USA), a digital camera (3.3 megapixelQCOLOR3C, QImagingTM) and image acquisitionsoftware (Qcapture Pro 5.1, QImagingTM). After-wards, images were digitized (at 400 and 1000¥),captured by Image-Pro Plus Software 6.0 (MediaCybernetics). In each case, photomicrographs of1280 ¥ 1223 pixels were obtained from non-coincident and consecutive fields.

2.10 Statistical analysis

The results are presented as mean plus standard errorof the mean (SEM). Statistical significance of differ-ences between groups was detected by one-wayanalysis of variance (ANOVA) or one-way ANOVArepeated measures, followed by Newman–Keuls mul-tiple comparison test using GraphPad software (SanDiego, CA, USA). p-Values (p < 0.05) were consideredas indicative of significance.

3. Results

3.1 Mechanical hypersensitivity

The SNC injury produced significant (p < 0.001)development of mechanical hypersensitivity on theipsilateral paw of the operated mice from the 7th tothe 19th day after the surgical procedure when com-pared with the sham group (Fig. 1B).

The results presented in Fig. 1A show that LEDTwith doses of 2.5 and 4 J/cm2 significantly (p < 0.001)inhibited mechanical hypersensitivity induced bySNC, with analgesic effect lasting for 30 min and inhi-bition values of 49 � 6% (2.5 J/cm2) and 62 � 7%(4 J/cm2). Treatments with 1 J/cm2 and with LEDdevice turned off (off group) were not effective. As2.5 J/cm2 did not statistically differ in effect from4 J/cm2 (p > 0.05) and requires less irradiation time,which would be less time consuming and thereforepreferable in a clinical setting, all subsequent experi-ments were conducted with 2.5 J.

Additionally, the results depicted in Fig. 1B showthat chronic LEDT (2.5 J) significantly (p < 0.001)inhibited mechanical hypersensitivity induced by theSNC on all treatment days, with maximum inhibitionof 63 � 12% on the 10th day post-surgery (Fig. 1B).

Moreover, Supporting Information Figure S2a–ddemonstrate that the time course of LEDT effect uponmechanical hypersensitivity is extended after repeatedapplications, as it lasted for 1 h on post-operative day10 (p < 0.01), 13 (p < 0.01) and 16 (p < 0.05).

3.2 Cold hypersensitivity

The surgical procedure resulted in a significantdecrease in the reaction latency (p < 0.001) to pain-related behaviours (paw lifting, licking or shaking)with corresponding increase in total time of reaction(p < 0.001) in SNC group animals from day 7 to day 19post-surgery (SNC vs. sham group in Fig. 1C and D).However, LEDT did not significantly (p > 0.05) inhibitcold hypersensitivity induced by SNC.

3.3 Pro- and anti-inflammatory cytokine levels

Spinal cord and sciatic nerve of the animal groupswere collected for cytokine analyses on the 13th dayafter SNC, after 7 days of consecutive LED treatments,as LEDT optimum effects in behavioural tests wereobtained on day 13 after SNC. Data presented in Fig. 2demonstrate that SNC group animals, in comparisonto sham group, presented significant (p < 0.05)increased levels of pro-inflammatory cytokines TNF-a



and IL-1b, in both spinal cord and sciatic nerve(p < 0.05). However, the concentration of anti-inflammatory cytokine (IL-10) did not differ signifi-cantly (p > 0.05) among experimental groups, neitheron the sciatic nerve nor on the lumbar spinal cord(SNC vs. sham group in Fig. 2).

LEDT significantly (p < 0.05) diminished both sciaticnerve and lumbar spinal cord TNF-a level in compari-son to SNC group, with inhibition values of 66 � 9%and 77 � 5%, respectively (Fig. 2A and B), but did notaffect the levels of IL-1b neither in the spinal cord(Fig. 2D) nor in the sciatic nerve (Fig. 2C).

3.4 Motor functional recovery and lower hindleg skeletal muscle mass

The SNC injury induced significant (p < 0.01) devel-opment of functional impairment on the ipsilateral

paw of operated mice, with motor function beinggradually restored during the assessment period, i.e.,from day 7 to day 19 after surgical procedure (SNC vs.sham group in Fig. 3A and B). However, chronic LEDTwas not significantly (p > 0.05) effective in accelerat-ing this functional recovery.

Moreover, the SNC resulted in a significant(p < 0.001) decrease in the mass (weight) of the ani-mal’s right lower hind leg soleus and gastrocnemiusmuscles, as observed on the 21st day post-injury (SNCvs. sham group in Fig. 3C and D). However, LEDT didnot significantly (p > 0.05) revert this loss.

3.5 Histological and morphological analysis

Morphological analysis demonstrates that SNC-operated groups, in comparison to sham-operated


(LEDT) (950 nm, 2.5 J/cm2) on the levels of (A)

sciatic nerve tumour necrosis factor alpha

(TNF-a), (C) interleukin (IL)-1b (e), IL-10 and

L4-L5 spinal cord (B) TNF-a, (D) IL-1b, (f) IL-10.

Material was collected on the 13th day after

sciatic nerve crush (SNC). Each column repre-

sents the mean of eight animals, and the verti-

cal lines indicate the standard error of the

mean. *p < 0.05 when comparing SNC + LEDT

with the SNC group; #p < 0.05 when comparing

SNC versus sham groups. Statistics: one-way

ANOVA followed by Newman–Keuls multiple

comparison test.


(LEDT) (950 nm, 2.5 J/cm2) on (A) sciatic static

and (B) functional indices and on (C) soleus and

(D) gastrocnemius muscle weight. ‘B‘ corre-

sponds to evaluation before surgical proce-

dure. Each group represents the mean of 8

animals, and the vertical lines indicate the

standard error of the mean. ###p < 0.001 when

comparing sciatic nerve crush (SNC) versus

sham groups. Statistics: (A, B) one-way analysis

of variance (ANOVA) repeated measures fol-

lowed by Newman–Keuls multiple comparison

test; (C, D) one-way ANOVA followed by

Newman–Keuls multiple comparison test.



group, presented significantly (p < 0.05) lower densityof myelinated fibres (Fig. 4A), although with no sta-tistical difference in myelin sheath of remaining fibres(Fig. 4C). SNC injury also induced significant(p < 0.001) reduction of myelinated fibre areas withcorresponding significant increase (p < 0.001) in con-junctive tissue area (SNC vs. sham group in Fig. 4Band D). Chronic LEDT did not significantly (p > 0.05)alter any of the aforementioned morphological param-eters. Although LEDT increased (p > 0.05), the densityof myelinated fibres did not significantly increased(37 � 2%; Fig. 4A).

4. Discussion

For the first time, the present study demonstrates thatLEDT at a dose of 2.5 J/cm2 (1) effectively reducedmechanical hypersensitivity but not cold hypersensi-tivity induced by SNC in mice; (2) decreased spinalcord levels of sciatic nerve TNF-a but did not affect theIL-1b and IL-10 levels; and (3) failed to acceleratemotor functional recovery and morphological nerveregeneration.

4.1 Effect of LEDT upon mechanical andcold hypersensitivity

In phototherapy, the correct choice of irradiationparameters, involving the selection of the appropriatedosimetry and wavelength, is essential for obtaining atherapeutic effect (Enwemeka, 2009). In accordancewith the law of Arndt–Schultz, biostimulation occursat doses between 0.5 and 10 J/cm2 (O’Kane et al.,1994; Yu et al., 1997; Schindl et al., 2003), withoptimal dose between 0.5 and 4 J/cm2 (Túner, 2004).Although these studies have been conducted withlow-intensity laser, it is suggested that such mecha-nisms are universal for different types of phototherapywith low intensity, as is the case of LEDT (Vinck et al.,2006; Barolet, 2008).

With regard to another important parameter in pho-totherapy, the wavelength of 950 nm was selected forthis study due to its optimum tissue penetration(within the ‘therapeutic window’ for phototherapy,600–1200 nm) (Niemz, 2007). In this spectral range(600–1200 nm), at the cellular level, LEDT modulatesfibroblast proliferation and collagen synthesis, pro-motes angiogenesis, stimulates macrophages and lym-phocytes by improving mitochondria energymetabolism, and promotes the production of growthfactors (Eells et al., 2004; Desmet et al., 2006). In addi-tion, Vinck et al. (2005) demonstrated that LEDT atthe wavelength of 950 nm with a dose of 1.07 J/cm2,applied to a midpoint on the intact skin overlying thecourse of the sural nerve, lowered nerve conductionvelocity and augmented negative peak latency, result-ing in a reduced number of impulses per unit of time.These neurophysiological activities of LEDT at a wave-length of 950 nm on sensory nerve conduction suggestits possible pain relief effect. Further investigation ofthe analgesic effect of different wavelengths and doseswould help determine the best parameters to treatdifferent types of pain.

The SNC was chosen for this study as it is a well-characterized model for peripheral nerve regenerationand NP (Vogelaar et al., 2004). In this model, as theinjury interrupts the axons but preserves the endo-neurial tubes (Vogelaar et al., 2004; Bauder and Fer-guson, 2012), full axonal regeneration and functionalrecovery is naturally achieved within 3–4 weeks afterthe injury (Bridge et al., 1994; Vogelaar et al., 2004; deHeredia and Magoulas, 2012; Hoang et al., 2012), asindicated here by the SSI and SFI results (see the SNCgroup in Fig. 3). As the injured axons grow into thedistal stump and re-establish functional connectionswith the peripheral target organs (Udina et al., 2011),NP-like symptoms (mechanical and thermal hyper-

Figure 4 Effect of light-emitting diode therapy (LEDT) (950 nm,

2.5 J/cm2) on nerve fibres morphometric parameters. Each column rep-

resents the mean of six animals, and the vertical lines indicate the

standard error of the mean. #p < 0.05 and ###p < 0.001 when comparing

sciatic nerve crush (SNC) versus sham groups. Statistics: one-way

analysis of variance followed by Newman–Keuls multiple comparison

test.



sensitivity) develop (Coronel et al., 2008; Fukui et al.,2010; Hoang et al., 2012) as a result of complex inter-actions of the injured peripheral nerve fibres withactivated glia (Schwann cells) and recruited immunecells (Chattopadhyay et al., 2007). In line with theresults obtained in this study (see SNC group inFig. 1C and D), in general, the pain-related behaviournaturally subsides 15–28 days after the nerve crush(Filliatreau et al., 1994; Bischofs et al., 2004; Chatto-padhyay et al., 2007; Coronel et al., 2008; Fukui et al.,2010; Pavic et al., 2011; Hoang et al., 2012), althoughthere are reports of prolonged tactile allodynia thatpersists from 70 days (Vogelaar et al., 2004) up to 52weeks after the injury (Bester et al., 2000).

It is important to mention that although LEDTinhibited mechanical hypersensitivity in all treatmentdays, chronic treatment did not accelerate tactilethreshold recovery as SNC-LEDT animals’ withdrawalresponse returned to SNC group animal’ levels everysingle day after LEDT analgesic effect ceased (seeFigs. 1A and S2a–d). Moreover, it should be noted thatLEDT chronic treatment, with the selected dose of2.5 J/cm2, did not induce tolerance during the experi-mental period. This is relevant because the treatmentof NP that is not responsive to first-line therapies isoften limited to opioids, which can be effective acutely,but have decreased efficacy after chronic exposure,inducing tolerance (Largent-Milnes et al., 2008).Additionally, LEDT analgesic effect is not due to stressinduced by restrain during treatment (32 s in the caseof 2.5 J/cm2), as procedure with the device turned offdid not affect the animals sensitivity threshold (Fig. 1A– Group Off).

Another interesting observation that is related toLEDT possible clinical relevance is the ‘duration ofeffect’. As demonstrated herein, LEDT analgesic effectis dose-dependent, which means that even though2.5 J/cm2 resulted in short-lived analgesia, i.e., 30 minafter first treatment on post-operative day 7 and up to1 h on days 10, 13 and 16 (Supporting InformationFig. S2), higher doses may yield more long-lastingeffects. In addition, in a clinical setting, treatment doesnot commonly consist of single-point stimulation asapplied here for experimental purposes. For instance,in the study conducted by Costa et al. (2008), 8–10points were treated twice a week for 5–8 weeks, withan energy density of 7.5 J per point and all patientshave responded positively to treatment. Although theauthors did not mention the duration of effect, in 73%of the cases, a ‘good’ or a ‘very good’ analgesiceffect was obtained. Moreover, due to overall meta-bolic and pharmacokinetic differences between thespecies, ‘duration of effect’ may vary considerably. For

example, oral doses of gabapentin are normallyadministered three times a day as half-life varies from4.8 to 8.7 h (Rose and Kam, 2002), although in ratmodels of NP, gabapentin analgesic effect lasted onlyfor up to 2 h in the spinal nerve ligation model(30 mg/kg, i.p.) (Cidral-Filho et al., 2011) and in thespared nerve injury model (30 mg/kg, s.c.) (Folkessonet al., 2010). However, additional studies are neces-sary to confirm this hypothesis.

On the other hand, LEDT was not effective againstcold hypersensitivity (Fig. 1C and D). The reasons forthis selective effect are likely related to the fact that theneural mechanisms underlying cold and mechanicalhypersensitivity are different, with each modalityreflecting activation of different populations of recep-tors, neuronal fibres as well as pain pathways (Huanget al., 2004; Neziri et al., 2011). Thus, more studies arenecessary in order to determine the underlying neu-romechanisms of LEDT-induced analgesia.

4.2 Effect of LEDT on pro-inflammatorycytokines

It has been shown that after SNC injury, various celltypes are activated and recruited to the injury site,which include mast cells, macrophages, fibroblasts,neutrophils and Schwann cells. These cells releasepro-inflammatory cytokines along with other inflam-matory mediators, such as chemokines, bradykininand prostaglandins (Scholz and Woolf, 2007), whichcontribute not only to the maintenance of inflamma-tion but also to the development of pain (Kulmatyckiand Jamali, 2007).

Similar results to the ones obtained in the presentstudy were those of Oshima et al. (2011), whorecently demonstrated that LEDT (630 nm, 2 J/cm2

and 870 nm, 2.5 J/cm2) decreased the levels of TNF-abut also did not affect the expression of IL-1b in thecartilage and synovial tissue in a rabbit model ofosteoarthritis. Note that the infrared (870 nm) treat-ment dose is the same as that used in the presentstudy, i.e., 2.5 J/cm2. On the other hand, LEDT(880 nm), with a much higher dose (7.5 J/cm2), notonly reduced the expression of mRNA TNF-a but alsodecrease the levels of IL-1b at the site of calcanealtendinitis in rats (Xavier et al., 2010b). Once more,differences in dosage may account for the differencesin effect observed among studies.

It is worth mentioning that our results are the firstdemonstration of the influence of LEDT on TNF-alevels in the central nervous system (CNS). Thisdatum is relevant as the involvement of CNS pro-inflammatory cytokines in the initiation and mainte-



nance of NP is well-established, as several cytokinessuch as IL-1b and TNF-a are increased in the dorsalhorn after nerve lesion, contributing to nerve injurypain, presumably by altering synaptic transmission inthe CNS, including the spinal cord (Inoue, 2006; Guoet al., 2007).

Lastly, an important observation is that althoughLEDT chronic treatment at the analgesic dose of 2.5 J/cm2 decreased pro-inflammatory cytokines levels bothin the crush site (sciatic nerve) as well as in the spinalcord, it is unlikely that the analgesic action of LEDTacute treatment is due to its TNF-a activity. It is wellknown that TNF-a and IL-1b play a key role in thedevelopment and maintenance of pain after peripheralnerve injury (Verri et al., 2006; Scholz and Woolf,2007; Watkins et al., 2007); therefore, therapies thateffectively reduce the expression of these cytokinesmay also present analgesic activity. Nevertheless, eventhough LEDT-induced analgesia is observed within30 min post-treatment on day 1, there is no evidencethat the anti-TNF-a action is already effective atprotein level in less than 30 min. Further investigationis required to clarify the mechanisms involved in LEDTanalgesia.

4.3 Effect of LEDT on morphological and motorfunctional neuronal regeneration

LEDT’s lack of regenerative effects presented hereindiffers from literature results, in which LEDT (940 nm,4 J/cm2) has been proven to stimulate the repairprocess of the calcaneal tendon in rats (640 nm, 20 J/cm2) (Bastos et al., 2009; Casalechi et al., 2009), aswell as the morphofunctional recovery after SNC inrats (940 nm, 9.5 mW, 4 J/cm2) (Serafim et al., 2012).The apparent contradictory results are probably due tothe differences in doses, i.e., 20 and 4 J/cm2, which aremore intense than the 2.5 J/cm2 used in our study.

Thus, at the dose used herein, the effects observedappear to be related to an analgesic and anti-inflammatory action rather than to a regenerativeactivity of the therapy. Additional investigation withhigher irradiation doses is necessary in order toconfirm the hypothesis that LEDT’s regenerative effectrequires higher dosages than the ones necessary toinduce analgesia.

4.4 Conclusion

In conclusion, our results demonstrate, for the firsttime, that (1) LEDT (950 nm, 80 mW/cm2, 2.5 J/cm2)is effective against NP, inhibiting mechanical but notcold hypersensitivity induced by SNC in mice; (2) this

analgesic effect is dose-dependent and does not inducetolerance; (3) LEDT effectively decreased the levels ofpro-inflammatory cytokine TNF-a in the spinal cordand sciatic nerve after SNC; and lastly, (4) chronictreatment with the analgesic dose of 2.5 J/cm2 neitheraccelerate motor functional recovery nor morphologi-cal nerve regeneration after SNC injury in mice.

Our results obtained in laboratory rodents extendprevious data and suggest that LEDT might be aninteresting and inexpensive complement to the treat-ment of NP.

Author contributions

All authors participated in the experiments, discussed theresults and commented on the manuscript.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Morphometric analysis of the sciatic nerve: (a)distal sciatic nerve at 400 ¥ magnification; (b) quantificationof the area of myelinated fibres; (c) quantification of the areaof connective tissue; (d) distal sciatic nerve at 1000 ¥ mag-nification for quantitation of the area of myelin sheath; (e)quantification of the area of the myelin sheath is made bysubtracting the value of the black area in (f) of the area (g).The sections were stained with Cason and ‘red oil’.Figure S2. Effect of LEDT on hypersensitivity induced bythe SNC in mice. Time course of effect of LEDT (950 nm, 1,2.5 and 4 J/cm2) on mechanical hypersensitivity on day (a)10, (b) 13, (c) 16 and (d) 19 after SNC. Each group representsthe mean of eight animals, and the vertical lines indicate theS.E.M. *p < 0.05, **p < 0.01 and ***p < 0.001 when compar-ing SNC+LEDT with SNC group; ###p < 0.001 when compar-ing SNC versus sham groups. Statistics: at each time point:one-way ANOVA followed by Newman–Keuls multiple com-parison test.



light-emitting diode therapy induces analgesia and decreases spinal cord and sciatic nerve tumour...

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