High energy photobiomodulation therapy in the early days of injury improves sciatic nerve regeneration in mice

Introduction: Different studies have evaluated the effects of electrophysical agents on regeneration after peripheral nerve injury. Among them, the most used in clinical and experimental research is photobiomodulation therapy (PBMT). Objective: To analyze the effect of standard energy (16.8 J) of PBMT on peripheral nerve regeneration, applied at different periods after sciatic nerve injury in mice. Methods: Thirty male Swiss mice were divided into six groups: naive; sham; control; LLLT-01 (660 nm, 16.8 J of total energy emitted in 1 day); LLLT-04 (660 nm, 4.2 J per day, 16.8 J of total energy emitted in 4 days); LLLT-28, (660 nm, 0.6 J per day, 16.8 J of total energy emitted over 28 days). The animals were evaluated using thermal hyperalgesia, Sciatic Functional Index (SFI), and Static Sciatic Index (SSI). Data were obtained at baseline and after 7, 14, 21, and 28 days after surgery. Results: For the SFI and SSI, all groups showed significant differences compared to the control group, and the LLLT-04 group presented the best results among those receiving PBMT. In the assessment of thermal hyperalgesia, there was a significant difference in the 14th day of evaluation in the LLLT-04 group. Conclusion: The application of 16.8 J was useful in sciatic nerve regeneration with an improvement of hyperalgesia, with higher efficacy when applied in four days (4.2 J/day).


INTRODUCTION
Peripheral nervous system (PNS) injuries have an annual incidence of 3%, generating a cost of around US$150 billion in the United States. Although PNS injuries do not put the individual's life at risk, it affects its quality of life, as trauma results in motor and sensory disabilities [1][2][3] .
The sciatic nerve injury model is highlighted in preclinical research and demonstrates its effectiveness in assessing the regeneration of the PNS, with this model different types Souza LG, Cardoso RB, Kuriki HU, Marcolino AM, Fonseca MCR, Barbosa RI ABCS Health Sci. 2020;45:e020016 of injury are reproduced. To assess PNS regeneration, the crush injury is the most used, as it maintains the connective support of the tissue, generating an injury to the axons, allowing the connectivity of the proximal and distal portion to the injury to be maintained, which favors Wallerian degeneration 4,5 . Different electrophysical agents have been used to improve the neural regenerative process. Among them we have the use of: ultrasound 6,7 , electrical stimulation 8 , and photobiomodulation therapy (PBMT), with emphasis on the use of low-level laser (LLL) [9][10][11][12][13][14][15] .
PBMT promotes the stimulation of microcirculation by paralysis of the pre-capillary sphincter, arteriolar and capillary vasodilation, vascular neoformation, which favor the increase of blood flow in the irradiated area 11 . It is also able to increase metabolism and cell proliferation, stimulating the production of adenosine triphosphate (ATP), triggered by the absorption of photons by cytochrome-c oxidase in the mitochondrial breathing chair [16][17][18][19] .
In addition, it has analgesic properties, which occur through the modulation of anti-inflammatory chemical mediators and synthesis of β-endorphin, which tends to limit the excitability of nociceptive receptors and eliminate algogenic substances 20-22 thus the effects of LLL contribute to the acceleration of the PNS regeneration process.
Although different studies demonstrate the efficacy of LLL therapy (LLLT) in nerve regeneration, there is still a large therapeutic window, using several protocols with different dosimetric parameters that can vary according to the wavelength, power, total energy emitted, density, duration, pulsed or continuous application and different points of application 23,24 .
The use of energy with known positive effects when applied in three to four weeks in previous studies, when concentrated in the first postoperative days may have photobiostimulatory or photobioinhibitory effects 25 .
Thus, the aim of this study was to analyze the effect of photobiomodulation (16.8 J) on peripheral nerve regeneration, applied in different regimes after sciatic nerve injury in mice. The mice were kept in cages, in groups of ten to twelve animals, in a controlled room temperature (22±2°C), with a light cycle divided into 12 hours of light, 12 hours of darkness, with free access to water and food. The animals were weighed and divided into six groups at random according to the procedures to be performed below: 1) Naive group (n=3): not submitted to the surgical procedure; and an area of 0.5 cm 2 was used, which makes the crushing process practical and reliable 10 . The crushing point was defined 5 mm above the three main branches (sural, fibular and tibial) and the device was maintained for 10 minutes at the crushing point.

METHODS
At the end, the nerve was relocated to its original bed and a suture of muscles and skin was performed (Tecnew TM , Quintino, Rio de Janeiro, Brazil) ( Figure 1).
The animals were physically contained manually and the low level   sensor, the time of 20 seconds was determined cut-off, in order to avoid possible tissue damage in the animals' paw 27,28 .
Three measurements of response time were performed, being recorded at 20-minute intervals in order to determine the baseline threshold, all groups were assessed before the surgical procedure. The evaluation of thermal hyperalgesia was performed on days 0, 14, 21, and 28 post-surgery.

Statistical analysis
The results were expressed as mean ± standard deviation.

RESULTS
The

Evaluation Sciatic Functional Index (SFI) and Sciatic Static Index (SSI)
Functional gait assessment provides the opportunity to assess specific aspects of sciatic nerve regeneration in a non-invasive The footprints were obtained preoperatively and at 7, 14, 21, and 28 days from the initial injury 31 .

Thermal Hyperalgesia
To assess thermal hyperalgesia, the Hargreaves ® device (Ugobasile, Comerio, Italy) was used. This emits an infrared light, which was radiated directly over the plantar region of the animal's right hind leg. The animals were housed in the test room one hour before the test. The paw withdrawal latency after the application of the thermal stimulus was automatically collected by means of a   Evaluation using thermal hyperalgesia to heat ( Figure 5) showed significant differences only in the LLLT-04 group on the 14 th day of the evaluation, which showed an increase in response time.

DISCUSSION
The crush injury model generates axonotmesis 35  In addition, Almeida et al. 40 evaluated biochemical changes induced by LLLT after axoniotymesis, in this study a wavelength of 660 and 808 nm was used for 21 days, providing a total energy of 12 J per day. It was observed that in the PBMT group there was an increase in sphingophospholipids and collagen, constituents of the myelin sheath, and also that the wavelength of 660 nm was more effective than 808 nm in relation to cell proliferation and PNS repair. In the present study, we can observe that the LLLT-04 and LLLT-28 groups did not show any differences when compared to the baseline, starting on the 21 st day of treatment, and that the LLLT-04 showed results closer to zero when compared to the other groups, suggesting that in 21 days the protocol was effective in treating nerve damage, in both groups with LLLT-04 being the most effective.
For the analysis of thermal hyperalgesia, measurements were not performed on the 7 th postoperative day, as a pilot study showed an increase in the incidence of surgical wound dehiscence during the handgrip. In the assessment of thermal hyperalgesia, differences were presented only for the LLLT-04 group on day 14, not following an improvement pattern for the groups.
In the research that related the use of PBMT and nerve damage, there is a large therapeutic window of the parameters used.
Thus, further studies are needed to verify the use of PBMT in early regeneration. The application of a high energy (J) in the first days after the traumatic injury appears to be a new perspective for treatment. Additionally, new pre-clinical and clinical studies are needed to verify functional restoration, improving the functional/sensory recovery process, in addition to speed in axonal regeneration.
From the above, it can be concluded that, in the sample analyzed, the PBMT protocol was effective in early nerve regeneration after sciatic nerve injury in mice, being more effective when the energy was applied during the initial four postoperative days (4.2 J/day).