Lasers in Medical Science

, Volume 27, Issue 3, pp 629–635

Low-level laser therapy improves repair following complete resection of the sciatic nerve in rats

Authors

  • Carla Christina Medalha
    • Department of BioscienceFederal University of São Paulo (UNIFESP)
  • Giuliana Castro Di Gangi
    • Department of BioscienceFederal University of São Paulo (UNIFESP)
  • Caroline Bublitz Barbosa
    • Department of BioscienceFederal University of São Paulo (UNIFESP)
  • Marcela Fernandes
    • Department of Orthopedics and TraumatologyFederal University of São Paulo (UNIFESP)
  • Odair Aguiar
    • Department of BioscienceFederal University of São Paulo (UNIFESP)
  • Flávio Faloppa
    • Department of Orthopedics and TraumatologyFederal University of São Paulo (UNIFESP)
  • Vilnei Mattioli Leite
    • Department of Orthopedics and TraumatologyFederal University of São Paulo (UNIFESP)
    • Department of BioscienceFederal University of São Paulo (UNIFESP)
Original Article

DOI: 10.1007/s10103-011-1008-9

Cite this article as:
Medalha, C.C., Di Gangi, G.C., Barbosa, C.B. et al. Lasers Med Sci (2012) 27: 629. doi:10.1007/s10103-011-1008-9

Abstract

The aim of this study is to analyze the effects of low-level laser therapy (LLLT) on the regeneration of the sciatic nerve in rats following a complete nerve resection. Male Wistar rats were divided into a control injury group, injury groups irradiated with a 660-nm laser at 10 or 50 J/cm2, and injury groups irradiated with an 808-nm laser at 10 or 50 J/cm2. Treatment began 24 h following nerve resection and continued for 15 days. Using the sciatic functional index (SFI), we show that the injured animals treated with 660 nm at 10 and 50 J/cm2 had better SFI values compared with the control injury and the 808-nm groups. Animals irradiated with the 808-nm laser at 50 J/cm2 show higher values for fiber density than do control animals. In addition, axon and fiber diameters were larger in animals irradiated with 660 nm at 50 J/cm2 compared to the control group. These findings indicate that 660-nm LLLT is able to provide functional gait recovery and leads to increases in fiber diameter following sciatic nerve resection.

Keywords

Laser therapyNerve regenerationSciatic nerve

Introduction

Lesions in the peripheral nerves are common injuries that result from the crushing or complete resection of the nerve, resulting in deterioration as well as the loss of sensitivity and motor function in the area innervated by the nerve [1]. The functional outcome of nerve repair is, in many cases, unsatisfactory [2]. A number of different repair methods have been under investigation, and advances have been made both in treatments to improve nerve regeneration as well as in improved methods to evaluate recovery [35].

One promising treatment is the use of laser light, which has been shown to effectively stimulate nerve function [6, 7]. However, a consensus regarding the use of this methodology, and standard protocols for its implementation, do not exist. In vitro studies have demonstrated that laser phototherapy can stimulate both the migration and fiber sprouting of neuronal cells, leading to the development of large neurons with densely branched and interconnected networks of processes [8]. In one such study, Barbosa et al. used a functional evaluation to assess the comparative effects of a 21-day treatment using low-power laser (660 and 830 nm) on sciatic nerve regeneration following crushing injuries [9]. The authors demonstrate that 660-nm laser irradiation is effective in promoting early functional recovery over the evaluation period, as indicated by the sciatic functional index (SFI). In addition, dos Reis et al. observed that a 660-nm laser, at 4 J/cm2, provides significant changes to myelin sheath area, but does not improve functional recovery following sciatic nerve injury in the rat [5].

In a clinical study, Rochkind et al. analyzed the effectiveness of low-power laser irradiation (780 nm) in the treatment of patients suffering from incomplete peripheral nerve damage [10]. They show statistically significant improvement in the recruitment of voluntary muscle activity in the laser-irradiated group, compared to the placebo group. In contrast, studies using a 904-nm laser found no statistically significant differences in the regeneration of crushed sciatic nerves [11]. Together, these findings suggest that laser phototherapy can progressively improve nerve function following injury, leading to significant functional recovery.

The mechanism via which LLLT influences nerve repair is not fully understood and, for many clinicians, the use of LLLT as a treatment modality remains controversial. Moreover, the laser energy delivered per unit area, defined as the fluence (J/m2), varies widely between studies, and this lack of standardized experimental conditions make it difficult to compare published results. Thus, before LLLT can be used as a standard clinical treatment, its safety and efficacy, as well as its effects at the cellular level, must be further assessed. In the current work, we sought to determine the impact of LLLT of varying fluence and wavelength upon sciatic nerve repair following a complete resection injury.

Materials and methods

This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of the Federal University of São Paulo (1465/09). Animals were maintained at 19–23°C on a 12:12 h light-dark cycle in the Animal Experimentation Laboratory of the Federal University of São Paulo. Rats were housed in plastic cages and had free access to water and standard food.

Fifty male Wistar rats (12 weeks old, ∼250 g) were divided into five experimental groups. All animals underwent the same surgical procedure, and were then weighed and randomly assigned to one of five treatment groups (ten animals per group) as follows: (1) sham-operated group, (2) 660-nm laser irradiation at 10 J/cm2, (3) 660-nm laser irradiation at 50 J/cm2, (4) 808-nm laser irradiation at 10 J/cm2 and, (5) 808-nm laser irradiation at 50 J/cm2.

Surgery procedures

Animals were anesthetized with a single intraperitoneal injection of a 1:4 combination of 10% ketamine (0.1 ml/100 g body weight) and 2% xylazine (0.07 ml/100 g body weight). Each animal was positioned in the ventral decubitus position, with its front and hind paws maintained in abduction. Antisepsis was carried out with iodine alcohol, followed by trichotomy and incision on the lateral face of the right thigh from the major trochanter to the knee. With the aid of a magnifying glass, the sciatic nerve was injured by a complete resection approximately 3 mm distal to the tendon of the internal obturator. An epineural anastomosis of the sciatic nerve was then performed with three simple sutures using nylon monofilament (Mononylon 10–0, Polysuture). The soft tissues were also sutured with nylon monofilament (Mononylon 10/0, Ethicon). Following surgery, each animal was given an injection of fentanyl (0.032 mg/kg), administered intraperitoneally for infection prophylaxis and analgesia. Analgesic was then administered at 12-h intervals for 2 consecutive days [12].

Laser irradiation

A low-energy continuous wave (cw) gallium-aluminum-arsenide (GaAlAs) laser (Teralaser, DMC Sao Carlos, São Paulo, Brazil) was used at 660 and 808 nm, with a 0.028-cm2 beam diameter, a power output of 30 mW at 10 J/cm2 (0.27 J per point, with irradiation time of 9 s) and 50 J/cm2 (1.41 J per point, with irradiation time of 47 s). The laser was applied to three points using the contact-point laser technique [13] and with the focus upon the damaged nerve area.

Laser irradiation was initiated 24 h following surgery and was then performed every 24 h using the following protocol: one session is defined as 5 consecutive days of irradiation with an interval of 2 days, and each animal completed 15 sessions. After the end of the treatment, animals were group-housed for an additional 4 months.

Sciatic functional index

Animal footprints were recorded to calculate the SFI. Animals were submitted to a walking track test with ink-painted paws. Each animal walked on a 43 × 8.7 cm tunnel, similar to that described by De Medinaceli et al. (1982) [14]. Footprints from all groups were obtained prior to surgery, and every 3 weeks thereafter for 4 months. The footprints on the paper strips were digitized, and parameter values were obtained using the ImageJ software package (NIH).

The Bain formula [15] was employed to calculate the SFI, as follows:
$$ \left[ { - {38}.{3} \times \left( {\left( {{\text{EPL}} - {\text{NPL}}} \right)/{\text{NPL}}} \right)\left] + \right[{1}0{9}.{5} \times \left( {\left( {{\text{ETS}} - {\text{NTS}}} \right)/{\text{NTS}}} \right)} \right] + {13}.{3} \times \left( {\left( {{\text{EIT}} - {\text{NIT}}/{\text{NIT}}} \right)} \right] - {8}.{8}; $$
where N was normal, E was experimental, SFI was the sciatic functional index, PL was the print length, TS was toe spread, and IT was intermediate toes (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-1008-9/MediaObjects/10103_2011_1008_Fig1_HTML.gif
Fig. 1

Schematic representation of the parameters assessed used to calculate the SFI. IT intermediate toes, PL print length, TS toe spread [9]

Euthanasia

Four months after surgery, all animals were deeply anesthetized with urethane (1.25 mg/kg) and sacrificed via transcardial perfusion with 100 ml isotonic saline at room temperature (containing heparin 0.2%), followed by 500 ml of fixative fluid (4°C) over a period of 20 min. The fixative consisted of 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer, pH 6.9.

Histology

After remotion of the distal sciatic nerve, it was soaked in 0.025 M sodium cacodylate buffer containing 25% glutaraldehyde (prefixed for 2 min and fixed in a refrigerator at 6°C for 12 h). Following fixation, the nerve segment was treated with 2% osmium tetroxide and 0.2% sodium cacodylate for 8 h. Segments were then rinsed in an isotonic sodium cacodylate buffer and dehydrated through a graded series of solutions of ethanol and propylene oxide. The segments were then embedded in a plastic resin (EPON 812) for 48 h at 60°C. The blocks were then cut transversely (0.5 μm thickness), beginning at the distal stump of the transected nerve sample, using a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with toluidine blue. Laminas were used for both quantitative and qualitative analyses.

Qualitative and histomorphometric analyses

Both qualitative and quantitative histological analyses were carried out by a pathologist who was blinded to the experimental group. For the qualitative analysis, a descriptive qualitative histopathological evaluation of the injured nerve area from each animal was carried out under a light microscope (Olympus, Optical Co. Ltd, Tokyo, Japan) at 40× magnification. Nerve segments were evaluated for alterations in tissue organization, fibrosis area, and the presence of myelinic and amyelinic fibers.

A DM4000B microscope equipped with a DFC320 digital camera, an IM50 image manager system, and a 63× oil-immersion objective (Leica, Germany) was used for the histomorphometric analysis of the injured area (previously identified in the histopathological observation). From each lamina, four microscopic images, measuring 350 × 350 μm, were randomly obtained (three images corresponding to the peripheral areas of injury, and the remaining image from the central area). The histomorphometric analysis was performed using the Image-Pro Plus software package (version 4.5.0.29). In each image, the myelin sheath area, the number of myelinated fibers, the axon diameter, myelin thickness, and fiber density were measured [16].

Statistical analysis

For histomorphometry, statistical comparisons of quantitative data were subjected to Kruskal–Wallis one-way analysis of variance (ANOVA) on rank test, followed by post-hoc Student–Newman–Keuls test. For the SFI statistical analysis, repeated-measures ANOVA was used, followed by Bonferroni's repeated-measures test. Statistical significance was established as p < 0.05.

Results

Sciatic functional index

Figure 2 shows the data of the functional gait analysis during the different experimental periods. As expected, the SFI values for all groups were significantly higher at the first, pre-injury, evaluation compared to the subsequent evaluations (p < 0.01). In addition, the SFI index of the 660-nm irradiated animals, at both energy intensities, was significantly greater than that measured for the other groups at the final evaluation (p < 0.05). No other differences were observed.
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-1008-9/MediaObjects/10103_2011_1008_Fig2_HTML.gif
Fig. 2

Mean SFI values for the groups studied. The asterisk refers to the difference between 660–10 and control and 660–50 and control

Qualitative histological analysis

The presence of both Wallerian degeneration, resulting from the discontinuity of axons, and degeneration of the distal stump are clearly detectable in all groups (Fig. 3). The presence of myelinated fibers with qualitative small caliber and thinner myelin sheath were found in all sectioned nerves (Fig. 3b, c, d, e and f) when compared to normal nerves (Fig. 3a). Sectioned axons presented with a disrupted morphology as compared to normal axons.
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-1008-9/MediaObjects/10103_2011_1008_Fig3_HTML.gif
Fig. 3

Photomicrograph of a semi-thin (2 μm) section cut transversely to the main axis of a normal sciatic nerve. a Sectioned/sutured control; b sectioned/sutured and treated with LLLT 660 nm at 10 J/cm2 and c 50 J/cm2; d LLLT at 808 nm and 10 J/cm2 and e 50 J/cm2. f 12 weeks post-injury. Scale bar = 20 μm. Magnification = 65×. Note the normal round shape of intact axons (*) versus the disrupted shape of sectioned axons (arrows)

Histomorphometry

Results of the morphometric analyses are reported in Table 1. Animals irradiated with the 808-nm laser, at 50 J/cm2, displayed a higher fiber density than the control group (p = 0.004). No other differences in fiber density were observed. Fiber diameters were wider in the groups irradiated with 660-nm laser, at both 10 J/cm2 (p = 0.02) and 50 J/cm2 (p = 0.01), compared to the control group. In addition, axon diameters were wider in animals irradiated with the 660-nm laser, at 50 J/cm2 (p = 0.01), compared to the control group. No statistically significant differences were observed in the analysis of fiber area. The myelin thickness/axon diameter ratio was reduced in animals treated with the 808 nm at both fluences, indicating less proportion between myelin thickness and axon diameter. No significant differences were observed between treated and non-treated nerves in myelin thickness or fiber/axon diameter ratio (p ≥ 0.1).
Table 1

Histomorphometric assessment of lesioned sciatic nerves treated with low-energy GaAlAs at 660 and 808 nm, both at 10 and 50 J/cm2

Groups

Density

Fiber diameter (D)

Axon diameter (d)

Fiber area (A)

Myelin thickness (D-d)/2

Myelin thickness/axon diameter ratio (D-d/2d)

Fiber/axon diameter ratio (D/d)

Control

5.289 ± 065

5.771 ± 0.14

2.845 ± 0.07

5.771 ± 0.33

1.442 ± 0.048

0.515 ± 0.01

2.03 ± 0.03

660-10

6.391 ± 0.44

6.854 ± 0.30*

3.176 ± 0.08

6.854 ± 0.40

1.548 ± 0.19

0.582 ± 0.11

2.164 ± 0.11

660-50

5.517 ± 0.59

7.466 ± 0.72*

4.104 ± 050*

7.466 ± 3.02

1.611 ± 0.08

0.428 ± 0.10

1.857 ± 0.10

808-10

3.782 ± 0.24

5.87 ± 0.55

3.241 ± 0.27

5.87 ± 1.43

1.286 ± 0.11

0.403 ± 0.03*

1.806 ± 0.03

808-50

11.976 ± 2.06*

6.12 ± 0.24

3.520 ± 0.19

6.12 ± 1.15

1.308 ± 0.03

0.372 ± 0.03*

1.744 ± 0.03

Discussion

The results of the current study reveal that laser therapy using a low-energy continuous wave laser at a wavelength of 660 nm produces partial gait recovery in injured animals following the complete resection of the sciatic nerve. Our data corroborate those of Belchior et al., who demonstrated functional recovery 20 days after sciatic nerve crush using 660-nm laser irradiation [3]. Similar results were found by Barbosa et al. using a 660-nm laser, at 10 J/cm2, on crushed nerve injuries [9].

In our current work, we performed morphological and morphometric analyses that revealed an increase in axon and fiber diameter in the injured animals receiving LLLT at 660 nm, with the largest effect occurring at a fluence of 50 J/cm2. No differences in recovery were observed in injured animals receiving LLLT at 808 nm, compared to control injured animals. These results are also in agreement with previous studies [9]. Recently, Gigo-Benato [2] suggested that 660-nm LLLT, at 10 or 60 J/cm2, accelerates neuromuscular recovery after nerve crush injury in rats. Therefore, the findings from the current study support the idea that functional gait improvements following 660-nm LLLT in injured animals may be a consequence of the improved histomorphometric parameters observed, such as axon and fiber diameter.

Interestingly, our morphometric analysis indicated that 808-nm LLLT induces significant increases in fiber density compared to untreated injured animals, likely as a result of injury-induced axonal sprouting. It is important to emphasize that this is a controversial issue in the literature. Some investigators state that increases in distal axonal sprouting may be a determinant of the success of regeneration and the specificity of reinnervation [16]. In contrast, other researchers have suggested that an increased number of regenerating axonal sprouts may be related to aberrant reinnervation and an impairment of functional recovery [17]. Currently, it is difficult to explain the increased fiber density observed following 808-nm LLLT, and to determine the implications this increase may have for nerve regeneration, particularly over a longer post-surgery period than that studied here.

In the current study, the qualitative analysis revealed that, in all groups, the nerve fascicles displayed a decrease in the presence of large-diameter myelinated fibers following injury. A significant number of small-diameter fibers were seen to be arranged in groups, suggesting axonal sprouting either in the endoneurial space (intrafascicular), extrafascicular space, or outside the surrounding fascicular borders. According to Ehrlicher et al., a focused laser spot can influence the actin-based processes necessary for growth cone extension in vitro [18]. Because these intracellular processes are not completely understood, it is difficult to fully explain the molecular basis of optical guidance. However, the authors hypothesize that optical forces may influence the diffusive behavior of globular proteins and small oligomeric structures that are free to move in the cytoplasm.

Our results support the ability of LLLT at specific wavelengths and energy fluences to influence nerve repair [19, 20]. The findings of this study are in agreement with those of Reis et al. and Belchior et al., who observed morphological and functional improvements in sciatic nerve-injured rats following 660-nm laser irradiation [3, 5]. In addition, Barbosa et al. found a positive effect of 660-nm laser irradiation on functional gait recovery in rats submitted to sciatic nerve injury, with no effect of 830 nm at the same fluence (10 J/cm2) [9]. These changes may represent a restorative response in the peripheral nerve that leads to improved functional recovery.

Our results also suggest that the effect of LLLT on tissues is fluence-dependent. We show that treatment with the 660-nm laser at the higher fluence (50 J/cm2) is more likely to produce morphological modifications in the injured nerve. Over a longer recovery period than that observed in the current study, these morphological alterations might lead to an improved functional gait recovery compared to treatment with the 660-nm laser at a lower fluence (10 J/cm2). It is important to highlight that this parameter has been highly variable in laser therapy studies related to nerve regeneration, with a wide range of fluences being used by different authors [3, 8].

Although the effects of LLLT have been demonstrated by a number of groups, the mechanisms underlying laser-induced alterations in tissues are poorly understood [20]. Photochemical signaling may be involved, in which laser light enhances cell proliferation via changes in mitochondrial physiology that subsequently affect RNA synthesis and alter the expression of various cell regulatory proteins. In addition, laser irradiation may influence the redox potential of target cells. For example, a shift in cellular redox potential toward oxidation is associated with the stimulation of cell functions, while a shift toward reduction may be inhibitory [21]. The differential effect of wavelength may be due to increases in ATP production following 660-nm laser irradiation that allow the cell a greater energy intake during the repair process [9]. However, further investigation is required to elucidate the mechanisms responsible for the stimulatory and inhibitory effects of laser irradiation on nerve repair.

Conclusions

Studies investigating the effects of different laser parameters on nerve repair are important to determine the efficiency and safety of laser irradiation and the treatment paradigms required for optimal stimulation. The present study examines the effects of different laser wavelengths and fluences on nerve injury repair. We demonstrate that different wavelengths produce different responses in the nerve tissue, and show a positive effect of 660-nm laser irradiation upon nerve repair. Further investigation is required to elucidate the mechanisms underlying the variable outcomes obtained using laser irradiation. Future studies will lead to a better understanding of the efficacy of laser therapy.

Copyright information

© Springer-Verlag London Ltd 2011