Effect of Laser Photobiomodulation with Gradual or Constant Doses in the Regeneration of Rats' Mental Nerve After Lesion by Compression

Abstract

Objective: Assess morphologically the efficacy of constant dose (CD) or gradual dose (GD) in photobiomodulation therapy (PBMT) during the regeneration process of rats' mental nerve after compression lesion. Materials and methods: Forty-eight male Wistar rats were used and divided into four groups (n = 12): negative control (NC): lesion by compression; positive control (PC): no lesion; GD: lesion by compression and PBMT with GD; and CD: lesion by compression and PBMT with CD. One day after the surgery, the groups GD and CD underwent PBMT daily in three equidistant points around the incision area. The parameters were wavelength of 808 nm, 100 mW, CD received treatment with 120 J/cm², while GD underwent the protocol of application: 1st and 4th sessions: 80 J/cm²; 5th to 8th sessions: 90 J/cm²; 9th to 12th sessions: 100 J/cm²; 13th to 16th sessions: 110 J/cm²; and 17th to 20th sessions: 120 J/cm². Euthanasias were performed at 3, 7, 14, and 21 days. Qualitative and quantitative analysis of the mental nerves were performed with ANOVA (analysis of variance) and Tukey tests (p ≤ 0.05). Results: It was observed that PBMT was able to accelerate the process of nerve regeneration presenting an increase in the number of myelinated fibers starting at 14 days of treatment for groups CD and GD, and at 21 days they were similar to PC. It was observed a better lamellar organization of myelin sheath at 7 days for GD and at 14 days for CD, similar to PC. Both GD and CD presented significant differences compared to NC and PC for thickness of the myelin sheath, outer perimeter, internal area, and number of myelin fibers. Conclusions: PBMT presented positive effect on the regeneration of nerve starting at 14 days, and after 21 days there was no difference between GD and CD.

Introduction

The mental nerve is one of the terminal branches of the inferior alveolar nerve, which emerges from the mandibular bone at the mental foramen and divides beneath the depressor anguli oris muscle into three branches as follows: one distributed to the skin of the chin and the other two to the skin and mucous membrane of the lower lip, being important for the sensitivity.¹ It can be injured during oral procedures such as apical surgery,^2,3 implant surgery,^4,5 and even at local anesthesia.⁶

The mental nerve injury can be solved spontaneously, but when that does not occur, the available therapeutic modalities present restricted results to certain cases, confirming that the best is prevention, and the good anatomy knowledge of the area by the dentist is of fundamental importance. Among the therapeutic modalities, the photobiomodulation therapy (PBMT) has been studied, with results indicating improved function, acceleration of regenerative process, reduce the inflammatory response in the nerve,⁷ metabolism increase in neurons, and better ability to produce myelin.⁸

Low-level diode lasers most widely studied in Dentistry have as active component the Gallium-Aluminum-Arsenate (GaAlAs).^7,9

–12 Using specific laser and light-emitting diode irradiation parameters, cellular activities can be induced, such as cellular proliferation and viability while stimulating mitochondrial activity, thereby increasing adenosine triphosphate production, synthesis of DNA and RNA, and activating cell-signaling cascades, including the production of reactive oxygen species, nitric oxide release, activating cytochrome c oxidase, and modifying intracellular organelle membrane activity, calcium flux, and expression of stress proteins.¹³ Due to its effects, PBMT is used in various procedures of oral surgery,¹⁴ periodontics,¹⁵ orthodontics,^16,17 pediatrics,¹⁸ and endodontics,¹⁹ among others.

In animal models, the use of PBMT resulted in stimulation of damaged fibers' axon growth.¹⁹ However, several different parameters are observed like 45 J irradiation for 10 days¹⁹; wavelength at 660 and 808 nm, with a 0.028-cm² beam diameter, power output of 30 mW at 10 J/cm² (0.27 J per point, irradiation for 9 sec), and 50 J/cm² (1.41 J per point, irradiation for 47 sec)¹⁰; energy density at the point: 10, 60, or 120 J/cm² with wavelength at 660 or 780 nm, spot area: 4 mm², power: 40 mW⁹; and wavelength 901 nm (impulsive) and power of 10 mW, in square-shaped window type device (16 cm²).⁸

Given the growing use of PBMT in dental offices and the search for a minimally invasive treatment for nerve injuries, this study aims to assess the regeneration of mental nerve after compression lesion and treatment with PBMT with different protocols.

Materials and Methods

Ethics considerations

This study was submitted to Ethics Committee for the use of animals of the Federal University of São Paulo, in which after analysis it was approved with the protocol number 213543.

Study groups

In this study, 48 Wistar rats with an average weight between 250 and 300 g were used. They were kept for 7 days housed in individual cages for acclimatization at 23°C room temperature with a 12-h light/12-h dark cycle.

The animals were randomly selected and divided into four study groups (n = 12). The groups NC (negative control—lesion by compression), GD (gradual dose—lesion by compression and PBMT with gradual), and CD (constant dose—lesion by compression and PBMT with CD) were submitted to lesion by compression of mental nerve; only animals of the PC (positive control) group were not submitted to injury. In the treatment phase, PC and NC groups composed the PC and NC groups, respectively, in which rats received no treatment with PBMT, only the laser pointer was positioned in the application points and not turned on; the animals of the treatment group CD received PBMT for 20 days, daily, starting 1 day after the surgery.

Surgery for compression of mental nerve

The surgical technique adopted in this study was based on Savignat et al.²⁰ and presented in Fig. 1. Under anesthesia with 2% xylazine (0.025 mL/100 g) and 10% ketamine hydrochloride (0.05 mL/100 g), submandibular incision was made to access the mental foramen. Then, the mental nerve was strongly crushed with an ultrafine forceps. The load for compression was standardized using the third ratchet of the ultrafine forceps for crushing the nerve for 15 sec. After surgery, the skin incision was sutured with a 3.0 Vicryl suture, and Betadine (Betadine, polyvidone iodee; VIATRIS, Merignac, France) was applied. The animals were maintained under analgesic (200 mg/L Paracetamol diluted in water) for 3 days and under antibiotic (Enrofloxacin—2.5 mg/kg) for 7 days.

FIG. 1.

Surgical technique used in this study. (a) Incision of the skin in the submandibular region; (b) dissection of the tissues and visualization of the mental nerve; (c) compression of the nerve with an ultrafine forceps; and (d) suture of the skin with a 3.0 Vicryl.

Treatment with PBMT

The PBMT device used was Whitening Lase II (DMC, São Carlos, Brazil), a low-power diode laser (GaAlAs). PBMT was performed in the animals of groups GD and CD in which were submitted daily during 20 days, starting 24 h after surgery. The application technique used was the punctual technique application for contact with tissue positioned perpendicularly to the skin at three equidistant points (with a distance of 1 cm between them) upon the suture. As showed on Table 1, for GD, parameters used were wavelength of 808 nm, 100 mW, and 22 sec (80 J/cm² at each point; 2.3 J energy for point), 25 sec (90 J/cm² at each point; 2.6 J energy for point), 28 sec (100 J/cm² at each point; 3.0 J energy for point), 31 sec (110 J/cm² at each point; 3.2 J energy for point), and 33 sec (120 J/cm² at each point; 3.5 J energy for point), with a spot area of 0.028 cm² and continuous mode of irradiation. For the CD group, parameters used were a wavelength of 808 nm, 100 mW, 33 sec (120 J/cm² at each point; 3.5 J energy for point) and spot area of 0.028 cm² with continuous irradiation.

Table 1.

Distribution of Study Groups According to the Treatment Protocols and Time Span Study

Study groups (n = 12)	Treatment protocols
NC	Lesion by compression
PC	No lesion by compression
GD	Lesion by compression+LLLT
	1st to 4th sessions: 80 J/cm², 2.3 J each point for 22 sec
	5th to 8th sessions: 90 J/cm², 2.6 J each point for 25 sec
	9th to 12th sessions: 100 J/cm², 3.0 J each point for 28 sec
	13th to 16th sessions: 110 J/cm², 3.2 J each point for 31 sec
	17th to 20th sessions: 120 J/cm², 3.5 J each point for 33sec
CD	Lesion by compression+LLLT (120 J/cm², 3.5 J each point for 33 sec)

CD, constant dose; GD, gradual dose; NC, negative control; PC, positive control.

Euthanasia

Three animals of all groups were euthanized at 3, 7, 14, and 21 days of study. The mental nerves were removed for analysis through transmission electron microscope (TEM).

TEM analysis

The collected mental nerves were prefixed in 4% PFA solution, postfixed in 1% osmium tetroxide, and included in spurr resin. Sections with 0.5 μm thickness were stained with toluidine blue, and images were acquired using AxioVision software (version 4.5). Ultrathin sections with 0.3 μm were collected on copper grids and contrasted in 5% uranyl acetate and 1% lead citrate and examined by TEM LEO 906E (Zeiss, Germany). Images of neural fibers were obtained with 20,000 × magnification (to count the number of myelinated fibers and thickness measurements of the myelin sheath, outer perimeter, and inner axonal diameter) and 100,000 × magnification (for evaluation of lamellar structure of myelin sheath). The measurements were performed using ImageJ software (version 1.47). The G-ratio was also evaluated, calculated by dividing the inner axonal diameter by the outer fiber diameter (that was composed by myelin sheath plus inner axonal diameter measurements), and the results were stratified in ranges of 0.4–0.5, 0.5–0.6, 0.6–0.7, 0.7–0.8, and 0.8–0.9. When ranges were used, the lowest portions were always included and the highest portions excluded (e.g., the 0–4 range includes 0 through 3.99, excluding 4). The results are expressed as means and considered significant values (p ≤ 0.05) after analysis of variance (ANOVA) and Tukey's test.

Results

The results were analyzed both qualitatively and quantitatively in a blind manner by two previously calibrated evaluators. The quantitative analysis was performed evaluating the number of myelinated fibers, thickness, outer perimeter, and inner area of the myelinated fibers in 20,000 × magnification photomicrographs. Qualitative analysis was performed with observations of the ultrastructure of rats' mental nerve with both 20,000 × and 100,000 × magnifications.

Qualitative analysis

For the ultrastructural analysis with 20,000 × magnification, it was observed that at 3 days the groups NC, CD, and GD presented large amount of myelinated fiber degeneration, which occurred at 7 days only in the group NC. At that same time, CD and GD already presented initial aspects of regeneration. At 14 days all groups showed increase of myelinated fibers regenerated. At 21 days, CD and GD reached a similar morphology to the PC group. Structures and organelles such as paranodes and macrophages were also observed (Fig. 2).

FIG. 2.

Cross-section TEM images of rats' mental nerve of groups PC, NC, GD, and CD on 3, 7, 14, and 21 days for ultrastructural analysis (20,000 × magnification). Black arrow, paranode; C, condensed connective tissue; CD, constant dose; d, axon degeneration; GD, gradual dose; m, macrophage; NC, negative control; PC, positive control; TEM, transmission electron microscope.

Ultrastructural morphologic analysis at 100,000 × magnification (Fig. 3) presented a better lamellar organization of myelin sheath at 7 days for GD and at 14 days for CD, similar to the patterns observed on PC. NC showed a large disorganization of lamellar structure in the first time span study, showing a similar pattern to PC only at 21 days. At the end of the treatment, there was no difference between CD or GD in this study.

FIG. 3.

Cross-section TEM images of rats' mental nerve of groups PC, NC, GD, and CD on 3, 7, 14, and 21 days for ultrastructural analysis of lamellae of the myelin sheaths (100,000 × magnification).

Quantitative analysis

The myelin sheath thickness of the CD group at 7, 14, and 21 days was similar to PC and varied over time (Table 2). For the outer perimeter (Table 3), GD presented similar value at 3 days compared to PC. The number of myelinated fibers (Table 4) showed a significant increase starting at 7 days of treatment for group CD, which achieved statistically similar mean values at 14 days compared to PC. The means of internal area (Table 5) differed according to the time span studies, presenting the lowest average at 3 days for the CD, GD, and NC groups. After, at 7 days, groups GD and CD were similar to PC, which occurred again only for CD at 21 days. The G-ratio values were calculated and presented in Table 6. NC, GD, and CD presented an increase since day 7 and CD showed statistically similar values compared to PC at 7, 14, and 21 days. The mean in groups NC and CD was lower at 3 days compared to PC and GD.

Table 2.

Mean Values of Thickness of the Myelin Sheaths in Function of Time Span and Study Groups

Days	NC	PC	GD	CD
3	0.223 (0.01)^Aa	0.683 (0.01)^Ba	0.698 (0.04)^Ba	0.328 (0.04)^Ca
7	0.421 (0.04)^Ab	0.611 (0.01)^Bb	0.519 (0.02)^Cb	0.658 (0.03)^Bb
14	0.345 (0.02)^Ac	0.512 (0.00)^Bc	0.313 (0.01)^Cc	0.456 (0.02)^Bc
21	0.411 (0.01)^Ab	0.627 (0.01)^Bb	0.471 (0.01)^Cb	0.635 (0.01)^Bb

Means followed by different letters (capital letters in horizontal and lower case letters in vertical) differ between them by ANOVA (p ≤ 0.05).

ANOVA, analysis of variance.

Table 3.

Mean Values of Outer Perimeter in Function of Time Span and Study Groups

Days	NC	PC	GD	CD
3	9.864 (0.20)^Aa	15.365 (0.05)^Ba	15.184 (0.05)^Ba	8.387 (0.06)^Ca
7	10.335 (0.07)^Aa	16.529 (0.28)^Bb	17.921 (0.03)^Cb	18.255 (0.16)^Cb
14	10.756 (0.09)^Aa	15.091 (0.02)^Ba	10.543 (0.09)^Ac	14.396 (0.07)^Cc
21	11.224 (0.07)^Ab	16.674 (0.15)^Bb	14.785 (0.13)^Cb	16.963 (0.21)^Bd

Means followed by different letters (capital letters in horizontal and lower case letters in vertical) differ between them by ANOVA (p ≤ 0.05).

Table 4.

Mean Values of Number of Myelin Fibers in Function of Time Span and Study Groups

Days	NC	PC	GD	CD
3	2^Aa	68^Ba	4.6^Ca	1.5^Ca
7	3^Aa	63.6^Bb	6.3^Ca	5.3^Cb
14	20^Ab	60.3^Bc	30^Cb	56.3^Bc
21	25^Ac	64.6^Bb	47.6^Cc	51.3^Bd

Means followed by different letters (capital letters in horizontal and lower case letters in vertical) differ between them by ANOVA and Tukey test (p ≤ 0.05).

Table 5.

Mean Values of Internal Area in Function of Time Span and Study Groups

Days	NC	PC	GD	CD
3	0.297 (0.002)^Aa	5.101 (0.07)^Ba	3.387 (0.03)^Ca	0.367 (0.01)^Da
7	3.847 (0.02)^Ab	6.633 (0.09)^Bb	6.61 (0.07)^Bb	6.592 (0.06)^Bb
14	3.667 (0.07)^Ab	5.871 (0.08)^Bc	3.295 (0.04)^Cc	5.774 (0.13)^Bc
21	5.423 (0.007)^Ac	5.777 (0.03)^Ac	6.591 (0.001)^Bb	5.862 (0.05)^Ac

Means followed by different letters (capital letters in horizontal and lower case letters in vertical) differ between them by ANOVA (p ≤ 0.05).

Table 6.

Mean Values of G-Ratio in Function of Time Span and Study Groups

Days	NC	PC	GD	CD
3	0.42^Aa	0.8^Ba	0.7^Ba	0.4^Aa
7	0.82^Ab	0.84^Ba	0.87^Cb	0.84^Bb
14	0.86^Ab	0.85^Ba	0.84^Cb	0.85^Bb
21	0.87^Ab	0.82^Ba	0.86^Cb	0.83^Bb

Means followed by different letters (capital letters in horizontal and lower case letters in vertical) differ between them by ANOVA (p < 0.05).

Discussion

Lesions in peripheral nerves such as mental nerve may occur at the clinical practice in procedures such as infiltrative anesthesia and implant surgery.^4,5,21 Lesion by compression, classified as axonotmesis, is one of the most frequent in clinical practice and also on in vivo animal model studies,^7,9,22 justifying the injury by compression applied in this study.

Studies report the use of PBMT on neurological treatments like spinal cord or brain lesions and peripheral nerve regeneration in animal models and clinical trials on humans.^23
–25 This study used the GaAlAs laser, which is the most common laser device in dental offices and the most studied for nerve regeneration.²²

In the present study it was observed the presence of macrophages, which act by removing inhibitory substances associated with myelin and indirectly release cytokines and mitogen factors to Schwann and fibroblast cells, stimulating cellular repair.²⁶ Such structure may be responsible for the regeneration observed in group NC, showing that the nerve can regenerate by itself, but with a slower healing process than when treated with PBMT.

As in this study, Medalha et al.¹⁰ also observed an increase in the density of myelinated fibers after laser irradiation of low level power with infrared spectrum (808 nm). Barbosa et al.²² also reported after 14 days of treatment, a significant improvement in locomotion after functional evaluation in rats. However, even in the NC group, it was also observed an increase in the number of myelinated fibers over time, corroborating with the results presented by Gigo-Benato et al.^9,27 It can be explained by the endogenous immunoglobulin antibody that assist in the removal of axons in degeneration and contribute to axonal regeneration after injury by promoting entry of macrophages and their phagocytic activity.²⁸

Differences in internal area measurements can be explained by concomitant increase in the number of myelinated fibers, thereby reducing the interaxonal space and compacting the myelinated fibers in the perineurium. Moreover, the thickness measurements of the myelin sheath and outer perimeter differed between the groups, with the CD group showing similar values compared to PC starting at 7 days, corroborating with results obtained by Cantín et al.²⁹ The authors stated that a very low dose (5 J/cm²) led to the increasing density of the myelin sheath, the same related by Gigo-Benato et al.⁹ that observed it with low and moderate doses.

For the outer perimeter, GD presented similar value at 3 days compared to PC, indicating that a low dose in the early stage of regeneration process may be indicated. According to Souza et al.,³⁰ the mitochondrial activity of macrophages of the activated cells irradiated with PBMT decreased after 1 day of culture. After 3 days of culture, irradiation exerted a positive modulation of the mitochondrial activity of macrophages, which might denote an increase in cell activation. After 5 days of culture, laser irradiation no longer modulated the mitochondrial activity of the activated cells. In our study we observed that GD presented similar values of outer perimeter compared to PC, probably because the lowest dose used in the beginning of the experiment was more efficient to induce activity of macrophages than the dose used in CD, resulting in a quicker regeneration process. However, Medalha et al.¹⁰ found that axon and fiber diameters were larger in animals irradiated with the highest dose compared to the control group. It can explain the differences between the groups CD and GD, in which the first one obtained higher values of myelin sheath thickness because it received the highest dose during all the experiment.

Our results indicated a positive effect of PBMT, both in CD and GD on mental nerve regeneration of rats after compression lesion, corroborating with several published studies.^{7,9
–11,22,27,29} Comparisons among studies that evaluated the PBMT effects for peripheral nerve regeneration becomes complex due to the variety of treatment protocols used. The duration of treatment ranged from 10 days,²⁷ 15 days,¹⁰ and 21 days.^7,11,22 The wavelength ranged among 660 and 808 nm,¹⁰ 660 and 830 nm,²² and 660 and 780 nm.⁹ In addition, the type of laser used varied: Nd:YAG,¹⁵ GaAlAs,²² ArGaAl,¹¹ and AlGaInP¹¹. Most of the studies assessed the sciatic nerve,^{7,9
–11,22,27,31} followed by the inferior alveolar nerve²⁹ and the mental nerve.²⁰ As observed, there are several different parameters used in the literature, so in this study it was adopted as the parameters recommended by the manufacturer of the PBMT device for both CD or GD.

Despite the lack of optimal G-ratio for the mental nerve in the literature, the theoretical studies of Rushton³² suggested that for a fixed outside diameter there should be an optimal myelin thickness for maximal conduction velocity. The arguments proposed that the G-ratio should have an optimal value at 0.6, maximizing conduction velocity, and that variation in G-ratio between 0.47 and 0.74 should result in a decrease of conduction velocity of not more than 5%. In addition, Chomiak and Hu ³³ showed that an optimal G-ratio for central nervous system is around 0.77 and for peripheral nerves is around 0.6. However, Hodgkin³⁴ used similar analytic arguments to arrive at an optimal value of 0.7.

Most of literature studies adopted a CD use of PBMT, which is followed by most of the clinicians worldwide for the treatment of neural injuries. However, Karu³⁵ suggested that the exposure effects to PBMT are cumulative and that its clinical effectiveness has an intrinsic relationship with adequate energy doses, if applied correctly, gradually, and regularly. This relationship is due to the capacity of low doses or overdose does not produce effects or even generate damages. The choice for a GD group was due to the possibility of occurrence of a neuroplasticity of the affected tissue when it is applied with CD for a long period, in a way that a stimulus that initially generated positive effects does not generate the desired effects later. Thus, our intention was to compare morphological aspects between GD and CD, in a way to relate to clinicians if CD is the best protocol to be followed or if a GD could provide better stimulation of the nerve and should be considered.

Within the limitations of this animal model in vivo study, it was possible to observe that PBMT presented a positive effect in the regeneration of the mental nerve. Therefore, more experimental studies are needed, as well as more parameter standardization with the use of PBMT for the regeneration of peripheral nerves.

Conclusions and Summary

In this animal model in vivo study, based on quantitative and qualitative analyzes, it can be concluded that PBMT presented a positive effect on nerve regeneration of rats' mental nerve from 14 days after injury, and at 21 days there was no ultrastructural morphologic difference between treatments with GD or CD.

Footnotes

Acknowledgment

This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Author Disclosure Statement

No competing financial interests exist.

References

Iwanaga

, Saga

, Tabira

, et al. The clinical anatomy of accessory mental nerves and foramina. Clin Anat, 2015; 28:848–856.

Concepcion

, Rankow

. Accessory branch of the mental nerve. J Endod, 2000; 26:619–620.

Arx

, Lozanoff

, Bosshardt

. Accessory mental foramina: anatomy and histology of neurovascularisation in four cases with apical surgery. Oral Surg, 2014; 7:216–227.

Kulkarni

, Kumar

, Kamath

, Thakur

. Accidental identification of accessory mental nerve and foramen during implant surgery. J Indian Soc Periodontol, 2011; 15:70–73.

Pancer

, Garaicoa-Pazmino

, Bashutski

. Accessory mandibular foramen during dental implant placement: case report and review of literature. Implant Dent, 2014; 23:116–124.

López

, Diago

. Failure of locoregional anesthesia in dental practice. Med Oral Patol Oral Cir Bucal, 2006; 11:e510–e513.

Camara

CNS

, Brito

MVH

, Silveira

, Silva

DSG

, Simões

VRF

, Pontes

WFP

. Histological analysis of low-intensity laser therapy effects in peripheral nerve regeneration in Wistar rats. Acta Cir Bras, 2011; 26:12–18.

Mohammed

, Al-Mustawfi

, Kaka

. Promotion of regenerative processes in injured peripheral nerve induced by low-level laser therapy. Photomed Laser Surg, 2007; 25:107–111.

Gigo-Benato

, Russo

, Tanaka

, Assis

, Salvini

, Parizotto

. Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med, 2010; 42:673–682.

10.

Medalha

, Di Gangi

, Barbosa

, Fernandes

, Aguiar

, Faloppa

. Low-level laser therapy improves repair following complete resection of the sciatic nerve in rats. Lasers Med Sci, 2012; 27:629–635.

11.

Shen

, Yang

, Liu

. Large-area irradiated low-level laser effect in a biodegradable nerve guide conduit on neural regeneration of peripheral nerve injury in rats. Injury, 2011; 42:803–813.

12.

Yan

, Chow

, Armati

. Inhibitory effects of visible 650-nm and infrared 808-nm laser irradiation on somatosensory and compound muscle action potentials in rat sciatic nerve: implications for laser-induced analgesia. J Peripher Nerv Syst, 2011; 16:130–135.

13.

Abrahamse

Regenerative medicine, stem cells, and low-level laser therapy: future directives. Photomed Laser Surg, 2012; 30:681–682.

14.

Freddo

, Giongo

, Ponzoni

, Corsetti

, Puricelli

Influence of a magnetic field and laser therapy on the quality of mandibular bone during distraction osteogenesis in rabbits. J Oral Maxillofac Surg, 2016; 74:2287.e1–2287.e8.

15.

Roncati

, Gariffo

. Systematic review of the adjunctive use of diode and Nd:YAG lasers for nonsurgical periodontal instrumentation. Photomed Laser Surg, 2014; 32:186–197.

16.

Torri

, Weber

. Influence of low-level laser therapy on the rate of orthodontic movement: a literature review. Photomed Laser Surg, 2013; 31:411–421.

17.

Carvalho-Lobato

, Garcia

, Kasem

, Ustrell-Torrent

, Tallón-Walton

, Manzanares-Céspedes

. Tooth movement in orthodontic treatment with low-level laser therapy: a systematic review of human and animal studies. Photomed Laser Surg, 2014; 32:302–309.

18.

Castro

, Abreu

, Correia

, Brasil

CMV

, Perez

DEC

, Pedrosa

FPR

. Low-level laser in prevention and treatment of oral mucositis in pediatric patients with acute lymphoblastic leukemia. Photomed Laser Surg, 2013; 31:613–618.

19.

Asnaashari

, Safavi

. Application of low level lasers in dentistry (endodontic). J Lasers Med Sci, 2013; 4:57–66.

20.

Savignat

, Vodouhe

, Ackermann

, Haikel

, Lavalle

, Libersa

. Evaluation of early nerve regeneration using a polymeric membrane functionalized with nerve growth factor (NGF) after a crush lesion of the rat mental nerve. J Oral Maxillofac Surg, 2008; 66:711–717.

21.

Hegedus

, Diecidue

. Trigeminal nerve injuries after mandibular implant placement—practical knowledge for clinicians. Int J Oral Maxillofac Implants, 2006; 21:111–116.

22.

Barbosa

, Marcolino

, de Jesus Guirro

, Mazzer

, Barbieri

, Fonseca

MCR

. Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers Med Sci, 2010; 25:423–430.

23.

Hashmi

, Huang

, Osmani

, Sharma

, Naeser

, Hamblin

. Role of low-level laser therapy in neurorehabilitation. PM R, 2010; 2(12 Suppl 2):S292–S305.

24.

Zecha

, Raber-Durlacher

, Nair

, et al. Low level laser therapy/photobiomodulation in the management of side effects of chemoradiation therapy in head and neck cancer: part 1: mechanisms of action, dosimetric, and safety considerations. Support Care Cancer, 2016; 24:2781–2792.

25.

Rochkind

Phototherapy in peripheral nerve regeneration: from basic science to clinical study. Neurosurg Focus, 2009; 26:E8.

26.

Madison

, Robinson

. Accuracy of regenerating motor neurons: influence of diffusion in denervated nerve. Neuroscience, 2014; 273:128–140.

27.

Gigo-Benato

, Geuna

, Rochkind

. Phototherapy for enhancing peripheral nerve: a review of the literature. Muscle Nerve, 2005; 31:694–701.

28.

Vargas

, Watanabe

, Singh

, Robinson

, Barres

. Endogenous antibodies promote rapid myelin clearance and effective axon regeneration after nerve injury. Proc Natl Acad Sci U S A, 2010; 107:11993–11998.

29.

Cantín

, Suazo

, Zavando

. Effect of low level laser on the perineural thickness of the inferior alveolar nerve. Int J Odontostomat, 2008; 2:123–127.

30.

Souza

, Ferrari

, Silva

, Nunes

, Bussadori

, Fernandes

. Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages. Braz J Phys Ther, 2014; 18:308–314.

31.

Rochkind

, Nissan

, Alon

, Shamir

, Salame

. Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats. Laser Surg Med, 2001; 28:216–219.

32.

Rushton

WAH.

A theory of the effects of fiber size in medullated nerve. J Physiol, 1951; 115:101–122.

33.

Chomiak

, Hu

. What is the optimal value of the G-ratio for myelinated fibers in the rat CNS? A theoretical approach. PLoS One, 2009; 4:e7754.

34.

Hodgkin

AL.

The Conduction of the Nerve Impulse. Springfield: Charles C. Thomas, 1964.

35.

Karu

TI.

Photobiological Fundaments of Low-Power Laser Therapy. London: Harwood Academic Publishers, 1989.