Effect of the laser therapy in association with swimming for a morphological nerve repair and functional recovery in rats submitted to sciatic axonotmesis

| The peripheral nerve injuries occur frequently and generally cause functional loss impacting negatively on patient’s life. The objective this study was to verify the efficiency of the combination of laser therapy and swimming in rats affected by axonotmesis. The sample was comprised of 50 Wistar rats and it was divided into 05 groups: Control Group; Surgical Control Group; Laser Experimental Group; Swimming Experimental Junior et al. Efeito da associação do laser e da natação no reparo nervoso 13 Group and Laser Experimental combined with Swimming Group. The nerve was crushed into a 5 mm-long segment next to the sciatic nerve trifurcation with a pair of forceps for 60 seconds. The GaAs infrared laser (904nm) was used with energy radiated 0,4J the first week, the second week 0,8J and 1,2J in the third and fourth week. For functional (FCI) evaluation, the animals were immobilized and the plantar region of their paws were painted with ink stamp. The procedure was repeated twice to each animal. The nerve morphometry (areas, diameters and thicknesses of the fibers, axons and myelin sheath) was performed with the measurement of 220 fibers per animal in each group . We can see that the GEL and GEN groups , obtained the best results when compared with the other groups (GC, GCC and GELAN) in all morphometric variables studied, but no statistically significant difference was found between them. In functional analysis, it was observed that the gelan group obtained the best results when compared with the other groups (GCC , GEN and GEL) and when the GEL and GEN groups were compared, there was no statistically significant difference between them. Was conclued the GEL and GEN groups havd the best morphometric results, while the GELAN showed the best functional outcome. Therefore, it can be concluded that the combination of these features favoured the functional recovery of the animals


INTRODUCTION
Peripheral nerve injuries are a common occurrence and generally cause the patient to suffer functional loss 1 that consequently has a negative effect on his/ her life 2 .The most common causes of peripheral nerve injury are firearm projectiles, falls and penetrative or contusion trauma, however, the main causative factor are automobile accidents 3 .
Peripheral nerve injury is more frequent in individuals aged between 25 and 40 years.This relatively young age may cause significant economic and social consequences due to functional disability occurring in early life, as most individuals are at the prime of their professional capacity during this period.Thus, following an injury, any treatment that leads to a more accelerated functional recovery of the peripheral nerves is extremely valuable to society 4 .The degenerative process in the muscle is known to start shortly after nerve injury, therefore, intervention to re-establish myoneural interaction must be swift 5 .
There are several surgical techniques and treatments that have been developed 6 , these help to regenerate the peripheral nerves with the aim of morphological and functional improvement in less time 7,8 .
Exercise is the preferred method out of the rehabilitation strategies that are currently used, mainly due to it being characterized as a non-invasive therapeutic resource 9 and that it represents an important mechanism for releasing neurotrophic factors, especially the mainly Brain-Derived Neurotrophic Factor (BDNF), which is considered paramount in terms of mediating neuroplasticity 10.11 .
Several authors have shown beneficial results by using exercise, namely through greater budding and extension of the axons 12 , increased number of myelinated nerve fibers, 13,14 and improved functional recovery of the injured member [15][16][17] .There have been extensive discussions in the scientific community 18 regarding recommendations as per the type of exercise as well as its duration and intensity.
Another feature used with the objective of promoting functional improvement for the injured person is phototherapy.Studies from the late 80s saw the beginning of thorough investigatory efforts whose purpose was to evaluate the protocols used as a therapeutic resource for regenerating peripheral nerves, these studies used crush injury 19 as an experimental model.In modern times these protocols are still used as a great therapeutic resource for peripheral nerve regeneration 20 .
Laser therapy, as an effective complementary therapeutic resource for treating nerve injuries, has gained prominence in physical therapy intervention protocols, which is mainly due to it being a noninvasive treatment that provides positive results for both regeneration and functional recovery.Among these benefits are: an anti-inflammatory and antioedematous effect, potential for wound healing, pain relief, increased mitochondrial respiration, ATP synthesis and proliferation of fibroblasts, stimulating the proliferation of Schwann cells that secrete neurotrophic factors for nerve regeneration, among other factors [21][22][23][24] .
Based on the belief in the beneficial effects of laser therapy and physical exercise, while considering the importance of functional recovery and returning to daily activities, the objective of this research was to investigate the effectiveness of laser therapy and swimming regarding the morphofunctional characteristics of rats that have been subjected to axonotmesis.It is our belief that the association of these protocols, in addition to the laser energy being gradually increased over the time of the injury, makes it possible to trigger a more efficient regenerative process that can provide the individual with more effective functional improvement.

METHODOLOGY
This study was approved by the Research Ethics Committee under the protocol number 034/2012.50 male, 80-day old, Wistar rats (Rattus norvegicus albinus) from the central bioterium were used.The animals were kept in cages and given ad libitum access to food and water in a controlled environment (temperature between 21 to 25°C with 12 hours in the light and 12 hours the dark) with no restrictions on movement.
The 50 animals were randomly divided in five groups (n=10, for each group) for axonotmesis, these being: Control group (CG), where the animals were not subject to any intervention and observed for 30 days.
Surgical Control Group (SCG), where the animals were put through the surgical intervention, with the induction of nerve injury through crushing, however, they were not subjected to any treatment protocol following the surgery.
Experimental Laser Group (ELG), in which the animals were put through the surgical intervention, with the induction of nerve injury through crushing, and, subsequent to surgery, were treated with the laser therapy protocol.
Experimental Swimming Group (ESG), in which the animals were put through the surgical intervention, with the induction of nerve injury through crushing, and, subsequent to surgery, were subjected to the swimming exercise protocol.
Experimental Laser together with Swimming Group (ELSG), in which the animals were put through the surgical intervention, with the induction of nerve injury through crushing, and, subsequent to surgery, were treated with laser therapy and subjected to the swimming exercise protocol.
In order to perform the surgical procedure on the nerve, the animals were anesthetized with a drug combination of Ketamine Hydrochloride (80 mg/kg) and Xylazine Hydrochloride (15 mg/kg).The surgical procedure involved a 5cm incision on the dorsolateral region of the left pelvic limb of the animal.Following the incision, the adjacent musculature was separated in order to expose the right sciatic nerve.
The nerve crushing was performed on a 5mm long segment of the sciatic nerve, close to the trifurcation where the fibular nerve, tibial nerve and sural nerve originate.The procedure was performed using a hemostat for a period of 60 seconds, comprising of three 20-second stages.The musculature and skin were sutured with 4-0 thread following the procedure.
The laser instrument used in the laser therapy groups was an AsGa-904nm (Arseneto de Gálio, ENDOPHOTON -KLD).The irradiation was performed on the skin of the animal's right pelvic limb, on the injured segment of the nerve.The procedures used by previously published studies with the same pattern were used 25,26 .The instrument was properly calibrated before the applications were performed.
The laser was applied, three times a week, for exactly 8 seconds in the first week, 16 seconds in the second week and 24 seconds in the third and fourth for a total treatment time of 4 weeks.The animals were held by hand during the application of the laser.
The following parameters used were: 904nm wavelength, 50mw power, 40J/cm² energy fluency or density in the first week, 80J/cm² in the second week and 120J/cm² in the third and fourth week, the radiated energy was 0.4J in the first week, 0.8J in the second week and 1.2J in the third and fourth week.The purpose of increasing the dose was to advance the stages of peripheral nerve injury from the acute to subacute phase, and later to the chronic phase.
For the groups being submitted to the swimming exercise, the animals were placed in a glass tank with warm water (32±2°C), filled to a 40cm depth.The swimming exercises were performed five times a week, with Saturdays and Sundays being rest days.During the first week, the animals swam exclusively for 20 minutes on the first day, with10 extra minutes being added each day up to a 40 minute duration, a time that was kept constant until the experiment's conclusion.
In order to evaluate the animals' functional capability, they were immobilized and placed in an acrylic pathway to walk on.The animals were filmed during their walk so that the footprints from their tracks could be recorded.This procedure was repeated with each animal twice.The distance between the footprints made by the hind limbs were evaluated according to the equation as described by Bain 27 , based on studies by De Medinacelli 28 .The videos were converted into sequential photos while following a calibration pattern for each image.The Sciatic Functional Index was used to evaluate functional recovery.The measurements were taken using 4.6.2Image Pro plus software, and the data were subjected to statistical analysis according to the P<0.05 index for all samples.
In order to collect the samples, the animals were anesthetized with a drug combination of Ketamine Hydrochloride (80 mg/kg) and Xylazine Hydrochloride (15 mg/kg), which was applied via intramuscular injection into the dorsolateral region of the left pelvic limb of the animal.Following the surgical sample collections, the animals were given a lethal dose of 150 mg/kg sodium pentobarbital and 2% lidocaine (20 mg/mL), which was administered intraperitoneally.
During the treatment, the histological samples from the sciatic nerve were fixed in Karnovsky solution, included in historesin and stained with osmium tetroxide.The samples were processed on a microtome in order to obtain the 5µm thick histological cuts.
The morphometry of the nerves was performed by measuring 220 fibers per animal in each group, which was done using a microcomputer with image capturing and analysis software linked to an optical microscope.The morphometric variables that were studied in the nerves are as follows: area of the fibers, area of the axons, minimum diameter of the fibers, minimum diameter of the axons, area of the myelin sheath and thickness of the myelin sheath.
The analysis of variance test (ANOVA) was used to compare the groups, followed by the TUKEY test when there was a significant difference detected.A paired T-test was used to make comparisons between the experimental and normal sciatic nerves.A significance level of p <0.05 was adopted for all analyses.

RESULTS
Upon comparing the area and the diameter of the nerve fiber, the ELG and ESG groups presented the best results (area = 38.42µm² and 37.56 µm 2 , respectively, and diameter = 4.02 µm and 3.96 µm, respectively) compared with the SCG and ELSG groups, which had 21.47 µm² and 28.76 µm² for area and 2.87 µm and 3.35 µm for diameter, respectively.The CG presented the highest mean for area with 50.12 µm² and 8.95 µm for diameter.
The data for the area and diameter of the nerve fibers can be seen in Table 1.Based on the results from Table 2, it is possible to see that the area and the diameter of the axon from the ELG and ESG groups had the best results in the two variables analyzed (area = 11.17µm² and 12.46µm², and diameter = 2.98µm and 3.06µm, respectively) when compared with the SCG and ELSG groups, which had 4.75µm² and 9.06µm 2 for the area of the axon and 2.05 µm and 2.57µm for the diameter of the axon.The CG showed the largest values with 15.05µm² of area and 5.1 µm of diameter.According to the results from Table 3, it is possible to see that the area and the thickness of the sheath from the ELG and ESG groups showed the best results (area = 25.25µm² and 25.10 µm², respectively; thickness = 2.86µm and 2.72µm, respectively) when compared with the SCG and ELSG groups, which showed 7.72m² and 21.70µm ² in the area of the sheath, and 1.82µm and 2.28µm in the thickness of the sheath, respectively.The CG presented the highest values with 43.07µm² in the area of the sheath and 3.82µm in the thickness of the sheath.Figure 1 shows the photomicrographs from the study groups and the morphological differences presented by these groups.Graphic 1 shows that the functional analysis results from the ELSG group were better than the other groups (SCG, ELG and ESG), with there being a statistically significant difference between them.The best functional analysis result is the one closest to the control group.Different letters indicate statistical difference (p<0.05).

Functional Values
Graphic 1. Graph showing the functional result of the groups.The ELSG Group showed a result closest to the CG The ELSG groups had a result of -44.90, which was the best due to it being closest to the CG result (-12.11).The ESG and ELG groups did not show any statistical difference, reaching -69.32 and -67.51, respectively.As a result of being denervated, the SCG group had the worst result (-82.81).

DISCUSSION
Several authors have chosen to work experimentally using crushing 29 when studying peripheral nerve injuries, which are classified by Seddon 30 as an axonotmesis.This method partly preserves important support structures, such as: the endoneurum, the perineurium and the tubules from the Schwann cells, which function to guide the new axon in regenerating up to the target organ.
Several experiments involving denervated animals 18 have described exercise as the factor that provides the most budding and extension of the axons, 12 the greatest increase in the number of myelinated fibers 13,14 and the best improvement in functional recovery for the injured member 15 .The results from this study are similar to those found in the literature regarding evaluating functional recovery, due to the fact that the groups that performed exercise showed better results compared to groups who did not go through the swimming protocol.Other authors refer to the harmless effects of physical exercise on the peripheral nerve regeneration process 31,32 .
Despite being somewhat controversial, some authors have suggested exercise as a complementary therapeutic resource 33 and, when combined with other therapeutic practices such as electrotherapy 34 or phototherapy 6,19 , can result in a better recovery prognosis for peripheral nerve injuries.The ELSG group in this research, which combined laser therapy with swimming, presented a better functional result when compared to the ELG and ESG group, respectively.
Based on clinical and experimental research, there is evidence that some of the effects of laser therapy are an improvement in nerve function, increased metabolism of the neurons and greater production capacity of the myelin sheath.Due to laser therapy not being invasive, the ability to radiate injured nerves without intervention is useful 35 .
Very little is known regarding the role of laser irradiation in terms of rehabilitating tissues of the locomotor system .However, laser irradiation is widely used to treat a variety of pathological conditions of the musculoskeletal system, including the peripheral nerves 25 .
Light energy absorption by nerve tissue increases ATP synthesis and cell proliferation, which increases axonal energy metabolism, improving scarring in the regenerative process, and thereby, the expression of neurotrophic factors, such as: GAP-43 protein, TGF-1, expression of the GCRP gene, which increase the regeneration rate and directs the axon to the target organ.The increase of axonal budding is also described as a result of the laser irradiation 42,43 .
During research performed by Reis 35 , the mean SFI (sciatic functional index) in the control group was -96.3, which is close to the figure found during this study, whose mean was of -82.81.The experimental laser group from the Reis research was different, with an average of -88.20 (-67.51 in our study).
According to Camargo 26 , who also used the AsGa laser on peripheral nerve regeneration, the mean SFI was -47.71, which represents an even better result compared with the data from this study.
During a study conducted by Endo 25 , who used low-level laser therapy to accelerate the regeneration of peripheral nerves , there was a progressive improvement of the SFI, both in irradiated and control nerves (69% and 45%, respectively).According to Endo 25 , the fiber density increased for the irradiated nerves and decreased for the control nerves, showing a significant difference between the two (p=0.001).The authors concluded that low-level laser therapy effectively accelerates sciatic nerve regeneration in rats.This study also presented an increase in the area of the fibers, with the experimental laser group and the surgical control group obtaining values of 38.42µm² and 21.47µm², respectively.
According to a study performed by Oliveira 31 , which involved using electrical stimulation and swimming for nerve regeneration and functional recovery during the acute phase of a axonotmesis in mice, the diameter of the axon was lower in the denervated groups, and that, when compared together, the group for which the best results were observed was the swimming group with the following values: 6.32±0.36 in the control group; 3.45±0.64 in the denervated group; 3.67±0.41 in the denervated + electro stimulation group; 4.34±0.69 in the denervated + swimming group; 4.04±0.38 in the denervated + swimming + electro stimulation group.Our study observed the same, the best result in relation to the diameter of the axon was in the swimming group.
During this study, the group in which there was an association between therapy and exercise, despite not showing good morphological results, presented an improvement in the functional analysis.We associate this improved functional result to the exercise, which requires the animal to release neurotrophic factors, and also to effect from the laser, which provides an antiinflammatory, anti-oedematous and analgesic effect.
We believe that further research should be conducted in order to identify the expression of proteins involved in the peripheral nerve regenerative process, as well as to verify the muscular response to newly received innervation, which establishes a correlation between the mioneural interaction and possible readaptation by the motor plates

CONCLUSION
Based on the results presented here, the laser and isolated swimming were observed to promote a morphological improvement during the evaluation of nerve regenerative process, however, no statistically significant difference between them was found.However, the association of laser therapy and swimming was beneficial for functional recovery following peripheral nerve injury.
Therefore, the conclusion is that laser therapy and swimming can promote the efficient morphological recovery of rats with peripheral nerve injury, and that associating these resources demonstrated a tendency for functional recovery.New swimming protocols should be therefore investigated with a view to establish a direct relationship between exercise intensity and functional recovery.

(Figure 1 .
Figure 1.Plate of the micrographs from the groups showing the morphology of the nerve (1000x)

Table 1 .
Mean values and standard deviation for the area of the nerve fibers (m²) and mean values and standard deviation of the smaller diameter of the nerve fibers (µm)

Table 2 .
Mean values and standard deviation of the area of the axons (m²) and mean values and standard deviation of the smallest diameter of the axons (µm) Different letters indicate a statistical difference (p <0.05)

Table 3 .
Mean values and standard deviation of the areas of myelin sheaths (m²) and mean values and standard deviation of the thicknesses of the myelin sheaths (µm) Different letters indicate a statistical difference (p <0.05)