Selective neural inhibition via photobiomodulation alleviates behavioral hypersensitivity associated with small sensory fiber activation.

OBJECTIVE: Photobiomodulation at higher irradiances has great potential as a pain-alleviating method that selectively inhibits small diameter nerve fibers and corresponding sensory experiences, such as nociception and heat sensation. The longevity and magnitude of these effects as a function of laser irradiation parameters at the nerve was explored. METHODS: In a rodent chronic pain model (spared nerve injury-SNI), light was applied directly at the sural nerve with four delivery schemes: two irradiance levels (7.64 and 2.55 W/cm2 ) for two durations each, corresponding to either 4.8 or 14.4 J total energy, and the effect on sensory hypersensitivities was evaluated. RESULTS: At emitter irradiances of 7.64 W/cm2 (for 240 s), 2.55 W/cm2 (for 720 s), and 7.64 W/cm2 (for 80 s) the heat hypersensitivity was relieved the day following photobiomodulation (PBM) treatment by 37 ± 8.1% (statistically significant, p < 0.001), 26% ± 6% (p = 0.072), and 28 ± 6.1% (statistically significant, p = 0.032), respectively, and all three treatments reduced the hypersensitivity over the course of the experiment (13 days) at a statistically significant level (mixed-design analysis of variance, p < 0.05). The increases in tissue temperature (5.3 ± 1.0 and 1.3 ± 0.4°C from 33.3°C for the higher and lower power densities, respectively) at the neural target were well below those typically associated with permanent action potential disruption. CONCLUSIONS: The data from this study support the use of direct PBM on nerves of interest to reduce sensitivities associated with small-diameter fiber activity.

Photobiomodulation and Vascularization in Conduit-Based Peripheral Nerve Repair: A Narrative Review.

Background: Peripheral nerve injuries pose a significant clinical issue for patients, especially in the most severe cases wherein complete transection (neurotmesis) results in total loss of sensory/motor function. Nerve guidance conduits (NGCs) are a common treatment option that protects and guides regenerating axons during recovery. However, treatment outcomes remain limited and often fail to achieve full reinnervation, especially in critically sized defects (>3 cm) where a lack of vascularization leads to neural necrosis. Conclusions: A multitreatment approach is, therefore, necessary to improve the efficacy of NGCs. Stimulating angiogenesis within NGCs can help alleviate oxygen deficiency through rapid inosculation with the host vasculature, whereas photobiomodulation therapy (PBMT) has demonstrated beneficial therapeutic effects on regenerating nerve cells and neovascularization. In this review, we discuss the current trends of NGCs, vascularization, and PBMT as treatments for peripheral nerve neurotmesis and highlight the need for a combinatorial approach to improve functional and clinical outcomes.

The Mechanistic Basis for Photobiomodulation Therapy of Neuropathic Pain by Near Infrared Laser Light

Background and Objective Various irradiances have been reported to be beneficial for the treatment of neuropathic pain with near infrared light. However, the mechanistic basis for the beneficial outcomes may vary based on the level of irradiance or fluence rate used. Using in vivo and in vitro experimentalmodels, this study determined the mechanistic basis of photobiomodulation therapy (PBMT) for the treatment of neuropathic pain using a high irradiance. Study Design/Materials and Methods ln vitro experiments: Cultured, rat DRG were randomly assigned to control or laser treatment (L T) groups with different irradiation times (2, 5, 30, 60 or 120s). The laser parameters were: output power » 960 mW, irradiance » 300mW/cm2, 808 nm wavelength and spot size » 3cm diameter/ area » 7.07cm2, with different fluences according to irradiation times. Mitochondrial metabolic activity was measured with the MTS assay. The DRG neurons were immunostained using a primary antibody to ß-Tubulin III. ln vivo experiments: spared nerve injury surgery (SNI), an animal model of persistent peripheral neuropathic pain, was used. The injured rats were randomly divided into three groups (n » 5). 1) Control: SNI without LT, 2) Short term: SNI with LT on day 7 and euthanized on day 7, 3) Long term: SNI with LT on day 7 and euthanized on day 22. An 808 nm wavelength laser was used for all treatment groups. Treatment was performed once on Day 7 post-surgery. The transcutaneous treatment parameters were: output power: 10 W, fluence rate: 270 mW/cm2, treatment time: 120s. The laser probe was moved along the course of the sciatic/sural nerve during the treatment. Within 1 hour of irradiation, behavior tests were performed to assess its immediate effect on sensory allodynia and hyperalgesia caused by SNI. Results ln vitro experiments: Mitochondrial metabolism was significantly lower compared with controls for all LT groups. Varicosities and undulations formed in neurites of DRG neurons with a cell body diameter 30µm or less. ln neurites of DRG neurons with a cell body diameter of greater than 30µm, varicosities formed only in the 120s group. ln vivo experiments: For heat hyperalgesia, there was a statistically significant reduction in sensitivity to the heat stimulus compared with the measurements done on day 7 prior to LT. A decrease in the sensitivity to the heat stimulus was found in the LT groups compared with the control group on day 15 and 21. For cold allodynia and mechanical hyperalgesia, a significant decrease in sensitivity to cold and pin prick was found within 1 hour after L T. Sensitivity to these stimuli returned to the control levels after 5 days post-L T. No significant difference was found in mechanical allodynia between control and L T groups for all time points examined. Conclusion These in vitro and in vivo studies indicate that treatment with an irradiance/fluence rate at 270 m W/cm2 or higher at the level of the nerve can rapidly block pain transmission. A combination therapy is proposed to treat neuropathic pain with initial high irradiance/fluence rates for fast pain relief, followed by low irradiance/ fluence rates for prolonged pain relief by altering chronic inflammation.

Low level laser therapy for the management of breast cancer-related lymphedema: A randomized controlled feasibility study.

OBJECTIVES: This study aimed to determine the feasibility of conducting a full scale randomized controlled trial investigating the effectiveness of low level laser therapy (LLLT), also known as photobiomodulation (PBM) therapy, used in addition to conventional therapy, for managing breast cancer related lymphedema (BCRL). MATERIALS AND METHODS: Patients with BCRL were recruited from the Southern District Health Board (New Zealand) via lymphedema therapists' referrals, and randomly allocated into either the laser group, which received BCRL conventional therapy (e.g., wearing compression garments, massage therapy, and/or exercise) plus a 6-week LLLT (PBM) intervention program (wavelength: 980/810 nm (80:20 ratio); output power: 500 mW beam spot size: 5 cm2 ; irradiance: 100 mW/cm2 ; treatment time per area: 1 minute dosage per area treated: 30J (6J/cm2 ); 10 points of treatment from axilla to wrist total LLLT (PBM) treatment time: 10 minutes total dosage delivered: 300 J), or the control group, which received BCRL conventional therapy alone. Feasibility was determined by recruitment and randomization rates, retention of participants and treatment protocol adherence, and was assessed during the recruiting and intervention periods. Data on participant satisfaction and adverse reactions of LLLT (PBM) were collected on completion of this study. Clinical outcomes (i.e., limb circumference, participant's perceived symptoms, psychological impacts, and activity disability) were assessed at baseline, and 6 and 12 weeks post-randomization. RESULTS: Over a 6-month recruitment window, 17 participants with BCRL were recruited in the study, and randomized into the two groups (recruitment rate of 81%, and randomization rate of 100%). Treatment adherence was high in the laser group (88.9% of participants completed all treatments). Retention rates were 88.9% for the laser group and 100% for the control group at both 6 and 12 weeks post-randomization. All participants who completed LLLT (PBM) treatment indicated that they were satisfied with the treatment. No serious adverse reactions were reported in this study. Clinical outcomes failed to show additional benefits of LLLT (PBM) intervention. CONCLUSION: This study demonstrated that it is feasible to conduct a fully powered RCT to definitively test the effectiveness of the additional use of LLLT (PBM) in the management of BCRL. For such a trial, 114 participants will be needed at baseline. Lasers Surg. Med. 50:924-932, 2018. © 2018 Wiley Periodicals, Inc.

Low level laser therapy (Photobiomodulation therapy) for breast cancer-related lymphedema: a systematic review.

BACKGROUND: Breast cancer related lymphedema (BCRL) is a prevalent complication secondary to cancer treatments which significantly impacts the physical and psychological health of breast cancer survivors. Previous research shows increasing use of low level laser therapy (LLLT), now commonly referred to as photobiomodulation (PBM) therapy, for BCRL. This systematic review evaluated the effectiveness of LLLT (PBM) in the management of BCRL. METHODS: Clinical trials were searched in PubMed, AMED, Web of Science, and China National Knowledge Infrastructure up to November 2016. Two reviewers independently assessed the methodological quality and adequacy of LLLT (PBM) in these clinical trials. Primary outcome measures were limb circumference/volume, and secondary outcomes included pain intensity and range of motion. Because data were clinically heterogeneous, best evidence synthesis was performed. RESULTS: Eleven clinical trials were identified, of which seven randomized controlled trials (RCTs) were chosen for analysis. Overall, the methodological quality of included RCTs was high, whereas the reporting of treatment parameters was poor. Results indicated that there is strong evidence (three high quality trials) showing LLLT (PBM) was more effective than sham treatment for limb circumference/volume reduction at a short-term follow-up. There is moderate evidence (one high quality trial) indicating that LLLT (PBM) was more effective than sham laser for short-term pain relief, and limited evidence (one low quality trial) that LLLT (PBM) was more effective than no treatment for decreasing limb swelling at short-term follow-up. CONCLUSIONS: Based upon the current systematic review, LLLT (PBM) may be considered an effective treatment approach for women with BCRL. Due to the limited numbers of published trials available, there is a clear need for well-designed high-quality trials in this area. The optimal treatment parameters for clinical application have yet to be elucidated.

The mechanistic basis for photobiomodulation therapy of neuropathic pain by near infrared laser light.

BACKGROUND AND OBJECTIVE: Various irradiances have been reported to be beneficial for the treatment of neuropathic pain with near infrared light. However, the mechanistic basis for the beneficial outcomes may vary based on the level of irradiance or fluence rate used. Using in vivo and in vitro experimental models, this study determined the mechanistic basis of photobiomodulation therapy (PBMT) for the treatment of neuropathic pain using a high irradiance. STUDY DESIGN/MATERIALS AND METHODS: In vitro experiments: Cultured, rat DRG were randomly assigned to control or laser treatment (LT) groups with different irradiation times (2, 5, 30, 60, or 120 seconds). The laser parameters were: output power = 960 mW, irradiance = 300 mW/cm2 , 808 nm wavelength, and spot size = 3 cm diameter/area = 7.07cm2 , with different fluences according to irradiation times. Mitochondrial metabolic activity was measured with the MTS assay. The DRG neurons were immunostained using a primary antibody to ß-Tubulin III. In vivo experiments: spared nerve injury surgery (SNI), an animal model of persistent peripheral neuropathic pain, was used. The injured rats were randomly divided into three groups (n = 5). (i) Control: SNI without LT; (ii) Short term: SNI with LT on day 7 and euthanized on day 7; (iii) Long term: SNI with LT on day 7 and euthanized on day 22. An 808 nm wavelength laser was used for all treatment groups. Treatment was performed once on day 7 post-surgery. The transcutaneous treatment parameters were: output power: 10 W, fluence rate: 270 mW/cm2 , treatment time: 120 seconds. The laser probe was moved along the course of the sciatic/sural nerve during the treatment. Within 1 hour of irradiation, behavior tests were performed to assess its immediate effect on sensory allodynia and hyperalgesia caused by SNI. RESULTS: In vitro experiments: Mitochondrial metabolism was significantly lower compared to controls for all LT groups. Varicosities and undulations formed in neurites of DRG neurons with a cell body diameter 30 µm or less. In neurites of DRG neurons with a cell body diameter of greater than 30 µm, varicosities formed only in the 120 seconds group. In vivo experiments: For heat hyperalgesia, there was a statistically significant reduction in sensitivity to the heat stimulus compared to the measurements done on day 7 prior to LT. A decrease in the sensitivity to the heat stimulus was found in the LT groups compared to the control group on days 15 and 21. For cold allodynia and mechanical hyperalgesia, a significant decrease in sensitivity to cold and pin prick was found within 1 hour after LT. Sensitivity to these stimuli returned to the control levels after 5 days post-LT. No significant difference was found in mechanical allodynia between control and LT groups for all time points examined. CONCLUSION: These in vitro and in vivo studies indicate that treatment with an irradiance/fluence rate at 270 mW/cm2 or higher at the level of the nerve can rapidly block pain transmission. A combination therapy is proposed to treat neuropathic pain with initial high irradiance/fluence rates for fast pain relief, followed by low irradiance/fluence rates for prolonged pain relief by altering chronic inflammation. Lasers Surg. Med. 49:516-524, 2017. © 2017 Wiley Periodicals, Inc.

Characterization of Macrophage/Microglial Activation and Effect of Photobiomodulation in the Spared Nerve Injury Model of Neuropathic Pain.

Objective: Neuropathic pain is common and debilitating with limited effective treatments. Macrophage/microglial activation along ascending somatosensory pathways following peripheral nerve injury facilitates neuropathic pain. However, polarization of macrophages/microglia in neuropathic pain is not well understood. Photobiomodulation treatment has been used to decrease neuropathic pain, has anti-inflammatory effects in spinal injury and wound healing models, and modulates microglial polarization in vitro. Our aim was to characterize macrophage/microglia response after peripheral nerve injury and modulate the response with photobiomodulation. Methods: Adult male Sprague-Dawley rats were randomly assigned to sham (N = 13), spared nerve injury (N = 13), or injury + photobiomodulation treatment groups (N = 7). Mechanical hypersensitivity was assessed with electronic von Frey. Photobiomodulation (980 nm) was applied to affected hind paw (output power 1 W, 20 s, 41cm above skin, power density 43.25 mW/cm 2 , dose 20 J), dorsal root ganglia (output power 4.5W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 85.5 J), and spinal cord regions (output power 1.5 W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 28.5 J) every other day from day 7-30 post-operatively. Immunohistochemistry characterized macrophage/microglial activation. Results: Injured groups demonstrated mechanical hypersensitivity 1-30 days post-operatively. Photobiomodulation-treated animals began to recover after two treatments; at day 26, mechanical sensitivity reached baseline. Peripheral nerve injury caused region-specific macrophages/microglia activation along spinothalamic and dorsal-column medial lemniscus pathways. A pro-inflammatory microglial marker was expressed in the spinal cord of injured rats compared to photobiomodulation-treated and sham group. Photobiomodulation-treated dorsal root ganglion macrophages expressed anti-inflammatory markers. Conclusion: Photobiomodulation effectively reduced mechanical hypersensitivity, potentially through modulating macrophage/microglial activation to an anti-inflammatory phenotype.

Comparison of Light Penetration of Continuous Wave 810 nm and Superpulsed 904 nm Wavelength Light in Anesthetized Rats.

OBJECTIVE: The purpose of this study was to investigate light transmission of continuous wave (CW) 810 nm wavelength light and 904 nm wavelength superpulsed light through skin and gastrocnemius muscle and skin only using an anesthetized Sprague-Dawley rat model. MATERIALS AND METHODS: The hair was shaved from the left thigh region of the anesthetized rats and a detector, which measured fluence rate, was placed either in the fascial plane deep into the muscle or below the dermis. The laser probe was placed in contact with the surface of the skin and measurements were taken for 4, 5, 10, 15, and 20 min depending on the experiment. RESULTS: The initial fluence rate measurements through the muscle and skin demonstrated that if the 904 nm wavelength superpulsed laser was turned on for a minimum of 15 min, there was no increase in light penetration over time. With appropriate warm-up periods, both lasers had stable output powers, which were reflected in stable fluence rate measurements over 4 min. The percentages of light transmission (fluence rate) through muscle and skin were 7.42% (810 nm wavelength) and 4.01% (904 nm wavelength) and through skin were 24.63% (810 nm wavelength) and 19.94% (904 nm wavelength). These data prove that transmission of CW 810 nm wavelength light through muscle and skin and skin alone is greater than transmission of superpulsed 904 nm wavelength light. CONCLUSIONS: It has been previously reported that superpulsing 904 nm wavelength light increased depth of penetration over time due to photobleaching. Based on our data, the observed increase in light penetration over time was due to an insufficient warm-up period of the superpulsed laser.

Photobiomodulation of the dorsal root ganglion for the treatment of low back pain: A pilot study.

BACKGROUND AND OBJECTIVE: Chronic low back pain is a worldwide public health issue with high socioeconomic impact. The aim of this study was to determine the efficacy of laser irradiation of the dorsal root ganglion of the second lumbar spinal nerve for chronic axial low back pain compared to lidocaine injection and radiofrequency treatment. STUDY DESIGN/MATERIALS AND METHODS: Twenty-eight patients were randomly divided into three treatment groups: lidocaine injection, radiofrequency, or laser. The second intervertebral foramen between the second and third lumbar vertebrae was accessed by percutaneous needle puncture bilaterally, guided by fluoroscopy. In the local anesthetic group, injection of 1 ml lidocaine without epinephrine was applied through a 20-gauge (G20) Quincke tip spinal needle inserted in the second lumbar intervertebral foramen. In the radiofrequency group, the probe (150 mm long with a 5 mm active tip) was directed through a G20 needle placed in the second lumbar intervertebral foramen and neuromodulation was done with a radiofrequency of Cosman G4® in pulses of 20 ms with wash-out period of 480 ms, for 300 seconds at 42°C. A single treatment was used. In the laser treatment group, a continuous wave, 808 nm wavelength diode laser (Photon Lase III® DCM, Brazil), with an output power of 100 mW was used for a single treatment. An 18 gauge needle was placed in the second lumbar intervertebral foramen guided by fluoroscopy. Light was delivered through a 600 µm optical fiber placed in the G18 needle. The tip of the fiber extended 5 mm beyond the tip of the needle in the second lumbar intervertebral foramen. The beam spot size was 0.003 cm(2) , irradiance = 35W/cm(2) , exposure time = 84 seconds, energy density = 2800J/cm(2) , total energy was 8.4 J. The low back pain score was assessed by the visual analog scale (VAS) and Pain Relief Scale (PRS) pre, post procedure and in 1 month follow up. Temperature was measured using a digital thermometer. RESULTS: All patients in the local anesthetic and laser treatment groups reported a pain reduction of at least 50% immediately post-procedure and 10 out of 11 patients in the radiofrequency group reported a pain reduction of at least 50%. At 1 month post-treatment, the laser treatment group had the greatest number of patients who reported more than 50% pain relief based on PRS (7 out of 10 patients) while only 2 out of 7 patients and 3 out of 11 patients in the lidocaine and radiofrequency treatment groups respectively reported more than a 50% pain relief. CONCLUSION: Laser irradiation caused an immediate decrease in low back pain post-procedure similar to pain reduction caused by lidocaine injection. Both lidocaine injection and laser irradiation were more effective than radiofrequency treatment for immediate and longer term (1 month post-treatment) chronic back pain. Lasers Surg. Med. 48:653-659, 2016. © 2016 Wiley Periodicals, Inc.

The antidepressant tranylcypromine alters cellular proliferation and migration in the adult goldfish brain.

The goldfish (Carassius auratus) is a widely studied vertebrate model organism for studying cell proliferation in the adult brain, and provide the experimental advantage of growing their body and brain throughout their ∼30-year life time. Cell proliferation occurs in the teleost brain in widespread proliferation zones. Increased cell proliferation in the brain has been linked to the actions of certain antidepressants, including tranylcypromine (TCP), which is used in the treatment of depression. We hypothesized that proliferation zones in the adult goldfish brain can be used to determine the antidepressant effects on cellular proliferation. Here, we report that bromodeoxyuridine (BrdU) labeling over a 24-hr period can be used to rapidly identify the proliferation zones throughout the goldfish brain, including the telencephalon, diencephalon, optic tectal lobes, cerebellum, and facial and vagal lobes. In the first 24 hr of BrdU administration, TCP caused an approximate and significant doubling of labeled cells in the combined brain regions examined, as detected by BrdU immunohistochemistry. TCP caused the greatest increase in cell proliferation in the cerebellum. The normal migratory paths of the proliferating cells within the cerebellum were not affected by TCP treatment. These results indicate that the goldfish provide significant advantages as a vertebrate model for rapidly investigating the effects of antidepressant drugs on cellular proliferation and migration in the normal and injured brain.

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.

Fibroblasts isolated from human middle turbinate mucosa cause neural progenitor cells to differentiate into glial lineage cells.

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for repair of spinal cord injury (SCI). Autologous transplantation of OECs has been reported in clinical trials. However, it is still controversial whether purified OECs or olfactory mucosa containing OECs, fibroblasts and other cells should be used for transplantation. OECs and fibroblasts were isolated from olfactory mucosa of the middle turbinate from seven patients. The percentage of OECs with p75(NTR+) and GFAP(+) ranged from 9.2% to 73.2%. Fibroblasts were purified and co-cultured with normal human neural progenitors (NHNPs). Based on immunocytochemical labeling, NHNPs were induced into glial lineage cells when they were co-cultured with the mucosal fibroblasts. These results demonstrate that OECs can be isolated from the mucosa of the middle turbinate bone as well as from the dorsal nasal septum and superior turbinates, which are the typical sites for harvesting OECs. Transplantation of olfactory mucosa containing fibroblasts into the central nervous system (CNS) needs to be further investigated before translation to clinical application.

808 nm wavelength light induces a dose-dependent alteration in microglial polarization and resultant microglial induced neurite growth.

BACKGROUND AND OBJECTIVE: Despite the success of using photobiomodulation (PBM), also known as low level light therapy, in promoting recovery after central nervous system (CNS) injury, the effect of PBM on microglia, the primary mediators of immune and inflammatory response in the CNS, remains unclear. Microglia exhibit a spectrum of responses to injury, with partial or full polarization into pro- and anti-inflammatory phenotypes. Pro-inflammatory (M1 or classically activated) microglia contribute to chronic inflammation and neuronal toxicity, while anti-inflammatory (M2 or alternatively activated) microglia play a role in wound healing and tissue repair; microglia can fall anywhere along this spectrum in response to stimulation. MATERIALS AND METHODS: The effect of PBM on microglial polarization therefore was investigated using colorimetric assays, immunocytochemistry, proteomic profiling and RT-PCR in vitro after exposure of primary microglia or BV2 microglial cell line to PBM of differing energy densities (0.2, 4, 10, and 30 J/cm(2) , 808 nm wavelength, 50 mW output power). RESULTS: PBM has a dose-dependent effect on the spectrum of microglial M1 and M2 polarization. Specifically, PBM with energy densities between 4 and 30 J/cm(2) induced expression of M1 markers in microglia. Markers of the M2 phenotype, including CD206 and TIMP1, were observed at lower energy densities of 0.2-10 J/cm(2) . In addition, co-culture of PBM or control-treated microglia with primary neuronal cultures demonstrated a dose-dependent effect of PBM on microglial-induced neuronal growth and neurite extension. CONCLUSION: These data suggest that the Arndt-Schulz law as applied to PBM for a specific bioassay does not hold true in cells with a spectrum of responses, and that PBM can alter microglial phenotype across this spectrum in a dose-dependent manner. These data are therefore of important relevance to not only therapies in the CNS but also to understanding of PBM effects and mechanisms.

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.

Pulsed light irradiation improves behavioral outcome in a rat model of chronic mild stress.

BACKGROUND AND OBJECTIVE: Transcranial laser therapy (TLT) has been used successfully for the treatment of stroke in animal models and clinical trials. These results support the hypothesis that TLT could be used to treat other central nervous system conditions, such as depression. Current therapy for depression emphasizes pharmaco-therapeutics. However, these interventions often cause unwanted side effects. Here, TLT as a treatment for depression was studied in a rat model of chronic mild stress (CMS). STUDY DESIGN/MATERIAL AND METHODS: Wistar rats were randomized into four experimental groups (n = 8): (1) No-stress; (2) stress without treatment (Stress); (3) stress treated with an antidepressant (Drug); and (4) stress treated with TLT (TLT). The rats in the stress groups were exposed sequentially to a variety of mild stressors for 8 weeks. Rats were weighed weekly. After 5 weeks of stressing, the Drug group received a daily injection of fluoxetine (10 mg/kg), and the TLT group was irradiated transcranially 3 times a week (810 nm wavelength laser, 3 mm diameter probe, 350 mW peak power, 100 Hz with 20% duty cycle, 2-minute treatment time, 120 J/cm(2) average energy density on skin surface). After 3 weeks of treatment, a forced swimming test (FST) was performed and recorded for behavioral assessment. Animals were euthanized after 8 weeks of the study. RESULTS: The No-stress group had significantly higher body weight than stress groups from week 5 (P < 0.05). No weight difference was found between the stress groups before treatment. However, the Drug group had significantly less body weight than both Stress and TLT groups after 2 weeks of treatment (P < 0.05). FST showed that the Stress group had significantly more immobility than the No-stress group (P < 0.05). Both Drug and TLT groups had significantly less immobility than the stress group (P < 0.05). There was no significant difference in immobility between both Drug and TLT groups (P = 0.62). CONCLUSIONS: TLT was comparable to fluoxetine in improving the behavioral outcome after CMS. TLT did not cause weight loss, which is consistently seen in patients treated with fluoxetine. This study demonstrates that TLT has potential as an effective treatment for depression.

Effect of 810 nm light on nerve regeneration after autograft repair of severely injured rat median nerve.

BACKGROUND AND OBJECTIVE: Destruction of large segments of peripheral nerves results in chronic loss of sensation and paralysis. For this type of severe injury, the defect can be bridged by nerve grafts. However, even with state-of-the-art microsurgical techniques, there is minimal recovery of sensation and motor function. Light therapy (LT) has been shown to improve functional outcome after surgical intervention to repair injured nerves using different techniques. Our objective was to investigate the effect of LT on peripheral nerve regeneration and function after severe median nerve injury and microsurgical autologous nerve graft repair using fibrin glue. STUDY DESIGN/MATERIALS AND METHODS: Adult female Sprague Dawley rats were used for this study. A 6-7 mm segment of the median nerve was excised and sural nerve segments from the same animal were used to bridge the gap using fibrin-based sealant. There were three experimental groups: control, autograft (AG), and autograft + LT (AG + LT). The AG + LT group received LT at the surgery site for 14 consecutive days using an 810 nm wavelength diode laser. Functional recovery was assessed bi-weekly by the grip strength test. Compound muscle action potential (CMAP) measurements were taken pre-injury and at 16 weeks post-surgery. Optical density measurement of S-100 immunoreactivity was done on the transplanted segment of the nerve. RESULTS: The AG + LT group had faster functional recovery of grip strength (P < 0.05), shorter CMAP latency (P < 0.05), and higher S-100 immunoreactivity (P = 0.0213) when compared to the AG group. However, at 15 weeks, grip strength in both the AG and AG + LT groups, while significantly improved, were still below control levels. CONCLUSION: These results suggest that LT can accelerate functional recovery and improve the quality of nerve regeneration after autograft repair of severely injured peripheral nerves.