Effects of systemic vascular photobiomodulation using LED or laser on sensory-motor recovery following a peripheral nerve injury in Wistar rats.

Peripheral nerve injury (PNI) is associated with considerable functional impairment. Photobiomodulation (PBM) has demonstrated positive effects regarding neuromuscular repair after PNI when applied locally to the nerve or injured muscle. However, the effects of systemic PBM with transcutaneous application over an important artery, which is also denominated vascular PBM (VPBM), remain unclear. The aim of the study was to compare the effects of VPBM with low-level laser (LLL) and light-emitting diode (LED) on gait, sensitivity and muscle morphology following a PNI. PNI was induced on Wistar rats using the sciatic nerve crushing technique. VPBM was performed over the rat's artery tail region with LED (850 nm, 40 mW, 3.2 J) and LLL (780 nm, 40 mW, 3.2 J). Gait functionality, mechanical (nociceptive) sensitivity, and morphology of the tibialis anterior muscle were evaluated at 7, 14, and 21 days after injury. An improvement in functional gait was shown in the VPBM-LLL group in all periods. Motor sensitivity was found after 14 days in the VPBM-LLL group. The left/right (L/R) muscle mass ratio revealed a reduction in muscle atrophy in the VPBM-LLL group at 7 days. Muscle fiber diameter increased in the VPBM-LED group at 14 days and increases in the cross-section area were found in the VPBM-LED and VPBM-LLL groups at 7 days. VPBM with both light sources (LED and LLL) positively modulated functioning and neuromuscular recovery following sciatic nerve injury in rats, with more pronounced results when using LLL.

Raman spectroscopy and sciatic functional index (SFI) after low-level laser therapy (LLLT) in a rat sciatic nerve crush injury model.

Axonotmesis causes sensorimotor and neurofunctional deficits, and its regeneration can occur slowly or not occur if not treated appropriately. Low-level laser therapy (LLLT) promotes nerve regeneration with the proliferation of myelinating Schwann cells to recover the myelin sheath and the production of glycoproteins for endoneurium reconstruction. This study aimed to evaluate the effects of LLLT on sciatic nerve regeneration after compression injury by means of the sciatic functional index (SFI) and Raman spectroscopy (RS). For this, 64 Wistar rats were divided into two groups according to the length of treatment: 14 days (n = 32) and 21 days (n = 32). These two groups were subdivided into four sub-groups of eight animals each (control 1; control 2; laser 660 nm; laser 808 nm). All animals had surgical exposure to the sciatic nerve, and only control 1 did not suffer nerve damage. To cause the lesion in the sciatic nerve, compression was applied with a Kelly clamp for 6 s. The evaluation of sensory deficit was performed by the painful exteroceptive sensitivity (PES) and neuromotor tests by the SFI. Laser 660 nm and laser 808 nm sub-groups were irradiated daily (100 mW, 40 s, energy density of 133 J/cm2). The sciatic nerve segment was removed for RS analysis. The animals showed accentuated sensory and neurofunctional deficit after injury and their rehabilitation occurred more effectively in the sub-groups treated with 660 nm laser. Control 2 sub-group did not obtain functional recovery of gait. The RS identified sphingolipids (718, 1065, and 1440 cm-1) and collagen (700, 852, 1004, 1270, and 1660 cm-1) as biomolecular characteristics of sciatic nerves. Principal component analysis revealed important differences among sub-groups and a directly proportional correlation with SFI, mainly in the sub-group laser 660 nm treated for 21 days. In the axonotmesis-type lesion model presented herein, the 660 nm laser was more efficient in neurofunctional recovery, and the Raman spectra of lipid and protein properties were attributed to the basic biochemical composition of the sciatic nerve.

Low-Level Laser Therapy in Different Wavelengths on the Tibialis Anterior Muscle of Wistar Rats After Nerve Compression Injury.

OBJECTIVE: Traumatic injuries are common and may promote disruption of neuromuscular communication, triggering phenomena that lead to nerve degeneration and affect muscle function. A laser accelerates tissue recovery; however, the parameters used are varied, making it difficult to compare studies. The purpose of this study was to evaluate the effect of low-level laser therapy, at 660- and 830-nm wavelengths, on the tibialis anterior muscle of Wistar rats after sciatic nerve compression. METHODS: Twenty animals were separated into 4 groups: control, sciatic nerve injury, lesion + 660-nm laser, and lesion + 830-nm laser. In the lesion groups, the right sciatic nerve was surgically exposed and compressed with hemostatic forceps for 30 seconds. After the third postoperative day, the groups with laser therapy were submitted to treatment for 2 weeks totaling 10 applications, performed directly on the surgical scar of the nerve injury. Grip strength was analyzed before and after the nerve injury and during the treatment period. The tibialis anterior muscle was processed for light microscopy, area measurement, smaller diameter, number of fibers, nuclei, and connective tissue. RESULTS: The animals submitted to the injury experienced muscular atrophy and morphological changes in the number of muscle fibers and nuclei. In the connective tissue morphometry, there was a decrease in the treated groups compared with the untreated groups. CONCLUSION: The laser treatment at different wavelengths showed no improvement in the tibialis anterior muscle of Wistar rats within the morphological and functional aspects evaluated.

Photobiomodulation in Neuroscience: A Summary of Personal Experience.

OBJECTIVE: This review summarizes personal experience with laser photobiomodulation and its potentials for the treatment of peripheral and central nerve system injuries. METHODS AND RESULTS: Laser photobiomodulation was shown to induce nerve cell activation, have a positive effect on metabolism of the nerve cells, and to stimulate nerve sprouting processes. Studies investigating the effects of laser photobiomodulation on injured peripheral nerves in rats reported immediate protective effects which increase the functional activity of the nerve, decrease or prevent scar tissue formation at the injured site, prevent or decrease degeneration in corresponding motor neurons of the spinal cord, and significantly increase axonal growth and myelinization. A direct application of laser on the spinal cord had a positive impact on the corresponding injured peripheral nerve and promoted recovery. A 780-nm laser phototherapy was applied following peripheral nerve reconstruction using a guiding nerve tube. Results showed myelinated axons crossing through the nerve tube and the continuation of axonal sprouting through the tube toward the distal part of the nerve. In a double-blind, placebo-controlled randomized pilot clinical trial in patients with incomplete stable long-term peripheral nerve injury (PNI), 780-nm laser irradiation progressively improved peripheral nerve function and led to substantial functional recovery. Muscle atrophy represents a major challenge in restorative medicine. Laser phototherapy was shown to increase biochemical activity and improve morphological recovery in muscle and, thus, could have a direct therapeutic application, especially during progressive muscle atrophy resulting from PNI. The effectiveness of composite implants of cultured embryonal nerve cells and the role of laser irradiation on regeneration and repair of the completely transected rat spinal cord were examined. Results suggested that laser photobiomodulation treatment accelerates the axonal growth. CONCLUSIONS: The significance of the performed experimental and clinical studies is in the provision of new laser technology in field of cell therapy and its therapeutic value for peripheral nerve and spinal cord injuries. Additional well-designed clinical studies are needed to evaluate the effectiveness and role of laser photobiomodulation treatment in a clinical setting.

Effects of photobiomodulation on experimental models of peripheral nerve injury.

Phototherapy has demonstrated positive effects in the treatment of peripheral nerve injury, but there is a need to investigate the dosimetric parameters. Thus, the aim of the present study was to conduct a literature review on the effects of photobiomodulation with the use of low-level laser therapy (LLLT) on the treatment of peripheral nerve injury in experimental models. The databases of PubMed/MEDLINE, SCOPUS, and SPIE Digital Library were searched for articles on the use of LLLT in experimental models of peripheral nerve injury published in English between January 2007 and March 2016. The laser parameter variability was wavelength (632.8 to 980 nm), power (10 to 190 mW), and total energy (0.15 to 90 J) in pulsed or continuous wave and single or multiple points. Eighteen original articles demonstrating the effects of LLLT on the acceleration of functional recovery, morphological aspects as well as the modulation of the expression inflammatory cytokines, and growth factors were selected. LLLT is a viable phototherapeutic modality for the treatment of peripheral nerve injury, demonstrating positive effects on the neuromuscular repair process using either red or infrared light. The majority of studies used a power of up to 50 mW and total energy of up to 15 J administered to multiple points. The determination of these parameters is important to the standardization of a LLLT protocol to enhance the regeneration process following a peripheral nerve injury.

X-ray irradiation has positive effects for the recovery of peripheral nerve injury maybe through the vascular smooth muscle contraction signaling pathway.

INTRODUCTION: It is well known that moderate to high doses of ionizing radiation have a toxic effect on the organism. However, there are few experimental studies on the mechanisms of LDR ionizing radiation on nerve regeneration after peripheral nerve injury. METHODS: We established the rats' peripheral nerve injury model via repaired Peripheral nerve injury nerve, vascular endothelial growth factor a and Growth associated protein-43 were detected from different treatment groups. We performed transcriptome sequencing focusing on investigating the differentially expressed genes and gene functions between the control group and 1Gy group. Sequencing was done by using high-throughput RNA-sequencing (RNA-seq) technologies. RESULTS: The results showed the 1Gy group to be the most effective promoting repair. RNA-sequencing identified 619 differently expressed genes between control and treated groups. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways. Among them, candidate genes associated with nerve repair were identified. DISCUSSION: Pathways involved in cell-substrate adhesion, vascular smooth muscle contraction and cell adhesion molecule signaling may be involved in recovery from peripheral nerve injury.

Photobiomodulation Triple Treatment in Peripheral Nerve Injury: Nerve and Muscle Response.

BACKGROUND: Muscle preservation or decrease in muscle degeneration and progressive atrophy are major challenges in patients with severe peripheral nerve injury (PNI). Considerable interest exists in the potential therapeutic value of laser phototherapy (photobiomodulation) for restoring denervated muscle atrophy and for enhancing regeneration of severely injured peripheral nerves. As previously published, the laser phototherapy has a protective and immediate effect in PNI. Laser phototherapy in the early stages of muscle atrophy may preserve the denervated muscle by maintaining creatinine kinase (CK) activity and the amount of acetylcholine receptor (AChR). OBJECTIVE AND METHODS: In the present study, the effectiveness of triple treatment laser phototherapy, namely, applied simultaneously at three areas: injured area of the peripheral nerve, corresponding segments of the spinal cord, and corresponding denervated muscle (triple treatment), was evaluated for the treatment of incomplete PNI in rats with the ultimate goal of achieving improved limb function. RESULTS: Forty-five days after the sciatic nerve insult, all rats regained normal walking (functional sciatic index values returned to baseline); however, the long laser irradiation (7 min) group presented the fastest recovery as opposed to short laser irradiation (3 min). A histological evaluation of the nerves revealed that long laser irradiation led to a higher amount of neuronal fibers that were larger than 4 µm (543 ± 76.8, p < 0.01) than short irradiation (283 ± 35.36). A histological evaluation of muscular atrophy showed that long laser irradiation evolved with significantly less muscle atrophy (8.06% ± 1.23%, p < 0.05) than short irradiation (24.44% ± 7.26%). CONCLUSIONS: The present study and our previous investigations showed that the laser phototherapy increases biochemical activity and improves morphological recovery in muscle and, thus, could have direct therapeutic applications on muscle, especially during progressive atrophy resulting from PNI.

Effect of low-level laser therapy (685 nm, 3 J/cm(2)) on functional recovery of the sciatic nerve in rats following crushing lesion.

Previous studies have shown that low-level laser therapy (LLLT) promotes posttraumatic nerve regeneration. The objective of the present study was to assess the efficacy of 685-nm LLLT at the dosage of 3 J/cm(2) in the functional recovery of the sciatic nerve in rats following crushing injury. The left sciatic nerves of 20 male Wistar rats were subjected to controlled crush injury by a hemostatic tweezers, and the rats were randomly allocated into two experimental groups as follows: control group and laser group. Laser irradiation (685 nm wavelength; 15 mW, CW, 3 J/cm(2), spot of 0.028 cm(2)) was started on the postsurgical first day, above the site of injury, and was continued for 21 consecutive days. Functional recovery was evaluated at 3 weeks postoperatively by measuring the sciatic functional index (SFI) and sciatic static index (SSI) at weekly intervals. The treated rats showed improvement in motion pattern. The SFI and SSI results were significant when comparing two groups on the 14th and 21st postoperative days (p < 0.05). There were intra-group differences detected in laser group in different periods (p < 0.05). Low-level laser irradiation, with the parameters used in the present study, accelerated and improved sciatic nerve function in rats after crushing injury.

In vitro and in vivo optimization of infrared laser treatment for injured peripheral nerves.

BACKGROUND AND OBJECTIVE: Repair of peripheral nerve injuries remains a major challenge in restorative medicine. Effective therapies that can be used in conjunction with surgical nerve repair to improve nerve regeneration and functional recovery are being actively investigated. It has been demonstrated by a number of peer reviewed publications that photobiomodulation (PBM) supports nerve regeneration, reinnervation of the denervated muscle, and functional recovery after peripheral nerve injury. However, a key issue in the use of PBM as a treatment for peripheral nerve injury is the lack of parameter optimization for any given wavelength. The objective of this study was to demonstrate that for a selected wavelength effective in vitro dosing parameters could be translated to effective in vivo parameters. MATERIALS AND METHODS: Comparison of infra-red (810 and 980 nm wavelengths) laser treatment parameters for injured peripheral nerves was done beginning with a series of in vitro experiments using primary human fibroblasts and primary rat cortical neurons. The primary rat cortical neurons were used for further optimization of energy density for 980 nm wavelength light using measurement of total neurite length as the bioassay. For these experiments, the parameters included a 1 W output power, power density of 10 mW/cm(2) , and energy densities of 0.01, 0.1, 0.5, 2, 10, 50, 200, 1,000, and 5,000 mJ/cm(2) . For translation of the in vitro data for use in vivo it was necessary to determine the transcutaneous penetration of 980 nm wavelength light to the level of the peroneal nerve. Two anesthetized, male White New Zealand rabbits were used for these experiments. The output power of the laser was set at 1.0 or 4.0 W. Power density measurements were taken at the surface of the skin, sub-dermally, and at the level of the nerve. Laser parameters used in the in vivo studies were calculated based on data from the in vitro studies and the light penetration measurements. For the in vivo experiments, a total of 22 White New Zealand rabbits (2.34-2.89 kg) were used. Translated dosing parameters were refined in a pilot study using a transection model of the peroneal nerve in rabbits. Output powers of 2 and 4 W were tested. For the final set of in vivo experiments, the same transection nerve injury model was used. An energy density of 10 mW/cm(2) at the level of the peroneal nerve was selected and the laser parameters were further refined. The dosing parameters used were: 1.5 W output power, 43 seconds exposure, 8 cm(2) area and a total energy of 65 J. RESULTS: In vitro, 980 nm wavelength light at 10 mW/cm(2) significantly improved neurite elongation at energy densities between 2 and 200 mJ/cm(2) . In vivo penetration of the infrared light measured in anesthetized rabbits showed that on average, 2.45% of the light applied to the skin reached the depth of the peroneal nerve. The in vivo pilot study data revealed that the 4 W parameters inhibited nerve regeneration while the 2 W parameters significantly improved axonal regrowth. For the final set of experiments, the irradiated group performed significantly better in the toe spread reflex test compared to the control group from week 7 post-injury, and the average length of motor endplates returned to uninjured levels. CONCLUSION: The results of this study demonstrate that treatment parameters can be determined initially using in vitro models and then translated to in vivo research and clinical practice. Furthermore, this study establishes that infrared light with optimized parameters promotes accelerated nerve regeneration and improved functional recovery in a surgically repaired peripheral nerve.

The use of shock waves in peripheral nerve regeneration: new perspectives?

Low-energy extracorporeal shock wave treatment (ESWT) is a relatively new therapeutic tool that is widely used for the treatment of epicondylitis and plantar fasciitis and to foster bone and wound healing. Shock waves, sonic pulses with high energy impact, are thought to induce biochemical changes within the targeted tissues through mechanotransduction. The biological effects of ESWT are manifested in improved vascularization, the local release of growth factors, and local anti-inflammatory effects, but the target cells too are influenced. ESWT appears to have differential effects on peripheral nerves and has been proved to promote axonal regeneration after axotomy. This review discusses the effects of ESWT on intact and injured peripheral nerves and suggests a multiple mechanism of action.

Effect of low-level laser therapy (LLLT) on acute neural recovery and inflammation-related gene expression after crush injury in rat sciatic nerve.

BACKGROUND AND OBJECTIVES: Peripheral nerve function can be debilitated by different kinds of injury. Low-level laser therapy (LLLT) has been used successfully during rehabilitation to stimulate recovery. The aim of this study was to evaluate the effects of LLLT (660 nm, 60 J/cm(2) , 40 mW/cm(2) ) on acute sciatic nerve injury. MATERIALS AND METHODS: Thirty Wistar male rats were divided into three groups: (1) Normal, intact nerves; (2) I3d, crushed nerves evaluated on Day-3 post-injury; (3) I + L3d, crushed nerves submitted to two sessions of LLLT and investigated at 3 days post-injury. Sciatic nerves were removed and processed for gene expression analysis (real-time PCR) of the pro-inflammatory factors TWEAK, Fn14 and TNF-α and extracellular matrix remodeling and axonal growth markers, such as TIMP-1, MMP-2, and MMP-9. Zymography was used to determine levels of MMP-2 and MMP-9 activity and Western blotting was used to evaluate TNF-α protein content. Shapiro-Wilk and Levene's tests were applied to evaluate data normality and homogeneity, respectively. One-way ANOVA followed by Tukey test was used for statistical analysis with a significance level set at 5%. RESULTS: An increase in TNF-α protein level was found in I + L3 compared to Normal and I3d (P < 0.05). Zymography showed an increase in proMMP-9 activity, in both I3d and I + L3d groups (P < 0.05). The increase was more evident in I + L3d (P = 0.02 compared to I3d). Active-MMP-9 isoform activity was increased in I + L3d compared to Normal and I3d groups (P < 0.05). Furthermore, the activity of active-MMP-2 isoform was increased in I3d and I + L3 (P < 0.05). An increase in TIMP-1 expression was observed in both I3d and I + L3d groups (P < 0.05). CONCLUSIONS: The current study showed that LLLT increased MMPs activity, mainly MMP-9, and TNF-α protein level during the acute phase of nerve injury, modulating inflammation. Based on these results, it is recommended that LLLT should be started as soon as possible after peripheral nerve injury.

Effects of low-level laser therapy after nerve reconstruction in rat denervated soleus muscle adaptation / Efeitos do laser de baixa potência após reconstrução nervosa na adaptação do músculo sóleo de rato

BACKGROUND: Peripheral nerve injury (PNI) rehabilitation remains a challenge for physical therapists because PNI effects are very disabling. Low-level laser therapy (LLLT) has been described as a physical resource that is able to influence enzymes called metallopeptidases (MMPs) associated with extracellular matrix (ECM) turnover, thus accelerating neuromuscular recovery after nerve crush injuries. However, the effects of LLLT in the treatment of severe nerve injuries and denervated slow-twitch muscles are still inconclusive. OBJECTIVES: The aim of this study was to evaluate the effects of different wavelengths and energy densities of LLLT irradiation, applied to a severe nerve injury after reconstruction, on denervated slow-twitch skeletal muscle adaptation. METHOD: Rats were submitted to a neurotmesis of the sciatic nerve followed by end-to-end neurorrhaphy. They received transcutaneous LLLT irradiation at the lesion site. The LLLT parameters were: wavelengths - 660 or 780 nm; energy densities - 10, 60 or 120 J/cm²; power - 40 mW; spot - 4 mm². Sciatic functional index (SFI), histological, morphometric, and zymographic analyses were performed. One-way ANOVA followed by Tukey's test was used (p<0.05). RESULTS: An atrophic pattern of muscle fibers was observed in all injured groups. The MMP activity in the soleus muscle reached normal levels. On the other hand, SFI remained below normality after PNI, indicating incapacity. No difference was found among PNI groups submitted or not to LLLT in any variable. CONCLUSIONS: LLLT applied to the nerve post-reconstruction was ineffective in delaying degenerative changes to the slow-twitch denervated muscles and in functional recovery in rats. New studies on recovery of denervated slow-twitch muscle are necessary to support clinical practice.

CONTEXTUALIZAÇÃO: A reabilitaçao das lesões nervosas periféricas (LNP) ainda é um desafio para a fisioterapia. A terapia com o laser de baixa potência (LBP) é descrita como um recurso físico capaz de interagir com enzimas relacionadas à alteração da matrix extracelular. Denominadas metalopeptidases (MMPs), essas enzimas atuam durante a recuperação neuromuscular após LNP. No entanto, os efeitos da LBP no tratamento de músculos desnervados de contração lenta após LNP graves ainda são inconclusivos. OBJETIVO: Avaliar os efeitos de diferentes comprimentos de onda e densidades de energia de irradiação de LBP, aplicado sobre o local do nervo após LNP grave e reconstrução. MÉTODO: Ratos foram submetidos a neurotmese do nervo isquiático e neurorrafia término-terminal. Os parâmetros do laser são: comprimento de onda: 660 ou 780 nm; densidades de energia: 10, 60 ou 120 J/cm²; potência: 40 mw; spot: 4 mm². O índice funcional isquiático (IFC) e análises histológicas, morfométricas e zimografia foram realizados. ANOVA one-way e teste de Tukey (p<0,05) foram utilizados. RESULTADOS: Um padrão atrófico das fibras musculares foi observado em todos os grupos com LNP. A atividade das MMPs no músculo sóleo alcançaram níveis normais. Entretanto, o IFC permaneceu inferior à normalidade após a LNP, indicando incapacidade. Não houve diferença entre os grupos de LNP submetidos ou não à LBP em qualquer variável. CONCLUSÃO: O LBP é incapaz de retardar alterações degenerativas em músculos sóleos desnervados e é ineficaz na recuperação funcional de ratos. Novos estudos sobre a recuperação do músculo de contração lenta desnervados são necessários para apoiar a prática clínica.

Effects of low-level laser therapy after nerve reconstruction in rat denervated soleus muscle adaptation.

BACKGROUND: Peripheral nerve injury (PNI) rehabilitation remains a challenge for physical therapists because PNI effects are very disabling. Low-level laser therapy (LLLT) has been described as a physical resource that is able to influence enzymes called metallopeptidases (MMPs) associated with extracellular matrix (ECM) turnover, thus accelerating neuromuscular recovery after nerve crush injuries. However, the effects of LLLT in the treatment of severe nerve injuries and denervated slow-twitch muscles are still inconclusive. OBJECTIVES: The aim of this study was to evaluate the effects of different wavelengths and energy densities of LLLT irradiation, applied to a severe nerve injury after reconstruction, on denervated slow-twitch skeletal muscle adaptation. METHOD: Rats were submitted to a neurotmesis of the sciatic nerve followed by end-to-end neurorrhaphy. They received transcutaneous LLLT irradiation at the lesion site. The LLLT parameters were: wavelengths--660 or 780 nm; energy densities--10, 60 or 120 J/cm²; power--40 mW; spot--4 mm². Sciatic functional index (SFI), histological, morphometric, and zymographic analyses were performed. One-way ANOVA followed by Tukey's test was used (p≤0.05). RESULTS: An atrophic pattern of muscle fibers was observed in all injured groups. The MMP activity in the soleus muscle reached normal levels. On the other hand, SFI remained below normality after PNI, indicating incapacity. No difference was found among PNI groups submitted or not to LLLT in any variable. CONCLUSIONS: LLLT applied to the nerve post-reconstruction was ineffective in delaying degenerative changes to the slow-twitch denervated muscles and in functional recovery in rats. New studies on recovery of denervated slow-twitch muscle are necessary to support clinical practice.