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Biphasic Dose Response in Low Level Light Therapy – An Update

Ying-Ying Huang, Sulbha K Sharma, Michael R Hamblin - Published online 2011 Sep 2. doi: 10.2203/dose-response.11-009.Hamblin (Publication)
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Low-level laser (light) therapy (LLLT) has been known since 1967 but still remains controversial due to incomplete understanding of the basic mechanisms and the selection of inappropriate dosimetric parameters that led to negative studies. The biphasic dose-response or Arndt-Schulz curve in LLLT has been shown both in vitro studies and in animal experiments. This review will provide an update to our previous () coverage of this topic. In vitro mediators of LLLT such as adenosine triphosphate (ATP) and mitochondrial membrane potential show biphasic patterns, while others such as mitochondrial reactive oxygen species show a triphasic dose-response with two distinct peaks. The Janus nature of reactive oxygen species (ROS) that may act as a beneficial signaling molecule at low concentrations and a harmful cytotoxic agent at high concentrations, may partly explain the observed responses in vivo. Transcranial LLLT for traumatic brain injury (TBI) in mice shows a distinct biphasic pattern with peaks in beneficial neurological effects observed when the number of treatments is varied, and when the energy density of an individual treatment is varied. Further understanding of the extent to which biphasic dose responses apply in LLLT will be necessary to optimize clinical treatments.

Keywords: low level laser therapy, photobiomodulation, biphasic dose response, reactive oxygen species, nitric oxide, traumatic brain injury

INTRODUCTION

Low level laser (light) therapy (LLLT) employs visible (generally red) or near-infrared light generated from a laser or light emitting diode (LED) system to treat diverse injuries or pathologies in humans or animals. The light is typically of narrow spectral width between 600nm – 1000nm. The fluence (energy density) used is generally between 1 and 20 J/cm2 while the irradiance (power density) can vary widely depending on the actual light source and spot size; values from 5 to 50 mW/cm2 are common for stimulation and healing, while much higher irradiances (up to W/cm2) can be used for nerve inhibition and pain relief. LLLT is typically used to promote tissue regeneration, reduce swelling and inflammation and relieve pain and is often applied to the injury for 30 seconds to a few minutes or so, a few times a week for several weeks. Unlike other medical laser procedures, LLLT is not an ablative or thermal mechanism, but rather a photochemical effect comparable to photosynthesis in plants whereby the light is absorbed and exerts a chemical change.

Within a decade of the introduction of LLLT in the 1970s it was realized that more does not necessarily mean better. The demonstration of the biphasic dose response curve in LLLT has been hampered by disagreement about exactly what constitutes a “dose”. Many practitioners concentrate on fluence as the principle metric of dose, while others prefer irradiance or illumination time. The use of very small spot sizes by some practitioners has led to the assertion that they delivered hundreds of mW/cm2 from a 50 mW laser. While this statement is mathematically correct it can give the impression that much higher doses of light were given than actually were delivered.

Two years ago we reviewed () the biphasic dose response in LLLT and found many reports in the literature concerning biphasic dose responses observed in cell cultures, some in animal experiments but no clinical reports. We now believe that the time is right to revisit this interesting topic for two reasons. Firstly because we have found more instances in our laboratory both in vitro with cultured cortical neurons, and in vivo with LLLT of traumatic brain injuries in mouse models. Secondly because advances have been made in mechanistic understanding of how LLLT works at a cellular level that may explain why a little light may be beneficial and at the same time a lot of light might be harmful.

MECHANISMS OF LOW LEVEL LIGHT THERAPY

Basic photobiophysics and photochemistry

According to the First Law of Photochemistry, the photons of light must be absorbed by some molecular photoacceptors or chromophores for photochemistry to occur ().The mechanism of LLLT at the cellular level has been attributed to the absorption of monochromatic visible and near infrared (NIR) radiation by components of the cellular respiratory chain (). Phototherapy is characterized by its ability to induce photobiological processes in cells. The effective tissue penetration of light and the specific wavelength of light absorbed by photoacceptors are two of the major parameters to be considered in light therapy. In tissue there is an “optical window” that runs approximately from 650 nm to 1200 nm where the effective tissue penetration of light is maximized. Therefore the use of LLLT in animals and patients almost exclusively involves red and near-infrared light (600–1100-nm) (). The action spectrum (a plot of biological effect against wavelength) shows which specific wavelengths of light are most effectively used for biological endpoints as well as for further investigations into cellular mechanisms of phototherapy (). Fluence (J/cm2) is often referred to as “dose”, though many authors and practitioners of LLLT also refer to energy (Joules) as dose. Not only is this confusing to the novice student of LLLT but it also assumes that the product of power and time (and more importantly power density and time) is the goal rather than the right combination of individual values. This lack of reciprocity has been shown many times before and since our first paper on biphasic dose response and several more authors have reported finding these effects since. Examples of recently published “dose-rate” effects are also reviewed later in this article.

Mitochondrial Respiration and Cytochrome c oxidase

Mitochondria play an important role in energy generation and metabolism and are involved in current research about the mechanism of LLLT effects. The absorption of monochromatic visible and NIR radiation by components of the cellular respiratory chain has been considered as the primary mechanism of LLLT at the cellular level (). Cytochrome c oxidase (Cco) is proposed to be the primary photoacceptor for the red-NIR light range in mammalian cells. Absorption spectra obtained for biological responses to light were found to be very similar to the absorption spectra of Cco in different oxidation states ().LLLT on isolated mitochondria increased proton electrochemical potential, ATP synthesis (), increased RNA and protein synthesis () and increases in oxygen consumption, mitochondrial membrane potential, and enhanced synthesis of NADH and ATP.

ROS release and Redox signaling pathway

Mitochondria are an important source of reactive oxygen species (ROS) within most mammalian cells. Mitochondrial ROS may act as a modulatable redox signal, reversibly affecting the activity of a range of functions in the mitochondria, cytosol and nucleus. ROS are very small molecules that include oxygen ions such as superoxide, free radicals such as hydroxyl radical, hydrogen peroxide, and organic peroxides. ROS are highly reactive with biological molecules such as proteins, nucleic acids and unsaturated lipids. ROS are also involved in the signaling pathways from mitochondria to nuclei. It is thought that cells have ROS or redox sensors whose function is to detect potentially harmful levels of ROS that may cause cell damage, and then induce expression of anti-oxidant defenses such as superoxide dismutase and catalase.

LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation () and increased ROS generation and cell redox activity have been demonstrated (). These cytosolic responses may in turn induce transcriptional changes. Several transcription factors are regulated by changes in cellular redox state, but the most important one is nuclear factor κB (NF-κB). Figure 1 graphically illustrates some of the intracellular signaling pathways that are proposed to occur after LLLT.

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Schematic depiction of the cellular signaling pathways triggered by LLLT. After photons are absorbed by chromophores in the mitochondria, respiration and ATP is increased but in addition signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO) are also produced.

NO release and NO signaling

There have been reports of the production and/or release of NO from cells after in vitro LLLT. It is possible that the delivery of low fluences of red/NIR light produces a small amount of NO from mitochondria by dissociation from intracellular stores (), such as nitrosothiols (), NO bound to hemoglobin or myoglobin (; ) or by dissociation of NO from Cco () as depicted in Figure 2. A second mechanism for NO production is by light-mediated increase of the nitrite reductase activity of cytochrome c oxidase (). A third possibility is that light can cause increase of the activity of an isoform of nitric oxide synthase (), possibly by increasing intracellular calcium levels. This low concentration of NO produced by illumination is proposed to be beneficial through cell-signaling pathways ().

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One possible theory that can explain the simultaneous increase in respiration an production of nitric oxide is the photodissociation of bound NO that is inhibiting cytochrome c oxidase by displacing oxygen.

BIPHASIC DOSE RESPONSES IN LLLT

Many reports of biphasic dose responses in LLLT were reviewed in our previous contribution and for convenience we have assembled these reports into Tables. Table 1 lists reports on cultured cells in vitro, Table 2 lists those reports in animal models in vivo, while Table 3 contains the only report of biphasic dose response in clinical studies.

TABLE 1.

Biphasic dose response studies of LLLT in vitro.

Year Cells Laser characteristics Fluence Irradiance Reference
1978 Lymphocytes in vitro   “threshold phenomenon”  
1990 Macrophage cell lines (U-937) 820nm Laser; 120mW/cm2; 2.4J/ cm2 to 9.6J/cm2 Cell proliferation: Maximum at 7.2J/cm2 least at 9.6J/cm2  
1991 Macrophage cell lines (U-937) 820nm Laser; 2.4J/cm2 or 7.2J/cm2; 400mW/ cm2 or 800mW/ cm2   cell proliferation increased at 400mW/ cm2; Cell viability reduced at 800mW/cm2
1994 Human oral mucosal fibroblast cells 812nm laser; 4.5mW/cm2; Cell proliferation peak at 0.45 J/cm2; less at 1.422J/cm2  
2001 Chinese hamster ovary and human fibroblast cells He-Ne laser;1.25 mW/cm2; 0.06 to 0.6J/cm2 Cell proliferation peak at 0.18 J/cm2; less at 0.6J/cm2.  
2003 human fibroblast cells 628nm LED; 11.46 mW/cm2; 0, 0.44, 0.88, 2.00, 4.40, and 8.68 J /cm2 Cell proliferation maximum at 0.88 J/cm2; reduced at 8.68 J/cm2  
2005 Human HEP-2 and murine L-929 cell lines 670 nm LED; 5 J/cm2 per treatment; Total 50J/cm2/day; 1 to 4 treatments/day Cell proliferation bigger at 2 treatments/day  
2005 Hela cells wavelength range of 580–860 nm DNA synthesis rate maximum at 0.1 J/cm2 with 0.8 mW/cm2  
2005 Wounded fibroblasts 632.8nm laser; 2mW/cm2; 0.5, 2.5, 5.0 or 10.0 J/cm2 Cell proliferation maximum at a single dose of 2.5J/cm2; Cellular damage at 10J/cm2  
2006 Wounded fibroblasts 632.8nm laser; 5.0 J/ cm2 or 16J/ cm2 Cell proliferation and cell viability increased at 5 J/cm2; decreased at 10 and 16 J/cm2  
2006 Wounded fibroblasts 632.8nm laser; 5.0 J/cm2 or 16J/cm2 Cell migration and proliferation increased at a single dose of 5.0 J/cm2 and two or three doses of 2.5 J/cm2; inhibited at 16 J/cm2  
2007 Human Neural Progenitor Cells (NHNPCs) 810nm; 0.2J/ cm2; 50mW/cm2 and 100mW/ cm2   Neurite outgrowth greater at 50mW/cm2; less at 100mW/cm2 Anders et al. 2007
2009 Rheumatoid arthritis synoviocytes 810nm laser_1, 3, 5, 10, 20 and 50 J/cm2 Cell proliferation increased at 5 J/cm2 (16.7 mW/cm2); Lower at 50 J/cm2  
2009 Mouse embryonic fibroblasts 810nm laser; 0.003,0.03,0.3,3 or 30J/cm2 NF-κB activation maximum at 0.3 J/cm2; decreased at 3 J/cm2 and 30 J/cm2  

TABLE 2.

Biphasic dose response studies of LLLT in vivo (animal models).

Year Tissue Laser characteristics Fluence Irradiance Reference
1979 wound closure He-Ne laser4 J/cm2   Wound healing best at 45 mW/cm2; least at 12.4 mW/cm2
2001 Induced heart attacks in rats 810 nm laser; 2.5 to 20mW/cm2 ;   Reductions of infarct size maximum at 5mW/cm2
Lower effects both at 2.5mW/cm2 and 20mW/cm2
2005 Mouse pleurisy induced by Carrageenan 650nm laser; 2.5 mW in 0.08 cm2; 3 J/cm2, 7.5 J/cm2, and 15 J/cm2 Inflammatory cell migration reduction most at 7.5 J/cm2; Less at 3 and 15 J/cm2  
2007 Healing of pressure ulcers in mice 670nm LED; 5 J/cm2 at 0.7, 2, 8 or 40mW/cm2   Healing significant improved only at 8mW/cm2;Less at 0.7, 2, and 40 mW/cm2
2007 Full thickness dorsal excisional wound in BALB/c mice a single exposure from 635, 670, 720 or 820nm filtered lamp; 1, 2, 10 and 50 J/cm2; 100 mW/cm2 10, 20, 100 and 500 seconds Healing effect best at 2 J/cm2 for 635nm light; worse at 50 J/cm2 for most wavelengths compared to no treatment 820nm was the best wavelength
2007 Inflammatory arthritis induced by zymosan in rats 810-nm laser; 3 and 30 J/cm2; 5 mW/cm2 and 50 mW/cm2 30 J/cm2 was better than 3 J/cm2 at 50mW/cm2 3 J/cm2 has effective at 5mW/cm2 but not 50mW/cm2

TABLE 3.

Biphasic dose response studies of LLLT in clinical studies.

Year Patients Laser characteristics Fluence Irradiance Reference
1997 Patients with post herpetic neuralgia of the facial type 830nm lasers; 60mW laser and 150mW laser; irradiance point at 4mm in diameter   Pain reduction greater at 150mW laser; less at 60mW laser when exposure to the same time.

Figure 3 shows a 3D depiction of the Arndt Schulz model to illustrate a possible dose “sweet spot” at the target tissue. This graph suggests that insufficient power density or too short a time will have no effect on the pathology, that too much power density and / or time may have inhibitory effects and that there may be an optimal balance between power density and time that produces a maximal beneficial effect. There even may be a (low) power density for which infinite irradiation time would only have positive effects and no inhibitory effect. We believe that the absolute figures will be different at different wavelengths, tissue types, redox states, and may be affected further by different pulse parameters.

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Three-dimensional model of the Arndt-Schulz curve illustrating how either irradiance or illumination time (fluence) can have biphasic dose response effects in LLLT.

CURRENT BIPHASIC DOSE RESPONSE STUDIES IN LLLT

In this section we cover the new reports of biphasic dose responses in LLLT that have been published in the last two years since our previous review.

In an oral mucositis hamster model Lopes and coworkers () delivered 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Both regimens delivered 0.9 J/cm2 per point. On day 7, 11 and 15 the authors reported reduced severity of clinical mucositis and lower levels of COX-2 staining in the 55 mW/cm2 group and that the 155 mW/cm2 had no significant differences when compared with controls. This data is summarized in Figure 4.

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Mean grading of oral mucositis (OM) in a hamster cheek pouch model treated with 0.9 J/cm2 of 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Graph redrawn from data contained in ().

Gal et al () compared the effects of delivering 5 J/cm2 of 670-nm laser at different power densities on wound tensile strength in a rat model. They found (Figure 5) that 670 nm laser achieved a significant effect using 4mW/cm2 applied for 1,250 seconds (20 mins 50 seconds) but that this effect was lost if the same 5J/cm2 fluence was delivered at 15 mW/cm2 for 333 seconds (5 mins 33 seconds).

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Mean wound tensile strength obtained after delivering 5 J/cm2 of 670-nm laser at different power densities (4mW/cm2 applied for 1,250 seconds or 15 mW/cm2 for 333 seconds). Graph redrawn from data contained in ().

() studied the effects of different influences of 980 nm laser on a human fibroblast in vitro model of wound healing. A small pipette was used to induce a wound in fibroblast cell cultures, which were exposed to a range of laser doses (1.5–66 J/cm2). Exposure to low- and medium-dose laser light accelerated cell growth, whereas high-intensity light negated the beneficial effects of laser exposure as shown in Figure 6.

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Mean percentage of healing induced in a scratch wounded culture of human fibroblasts using different fluences (constant time, increasing irradiance) of 980-nm laser. Graph redrawn from data contained in ().

() performed a dose response study by applying a 7 mW HeNe (632.8-nm) laser with a power density of 4 mW/cm2 to 15×15 mm excisional wounds on Swiss albino mice for a range of irradiation times from 249 seconds (4.15 mins) up to 2,290 seconds (41.46 mins). As Figure 7 shows, there was a clear biphasic response (including a possible inhibitory effect) with changes in irradiation time and therefore fluence.

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Mean area under the curve of wound area over time in a mouse excisional wound healing model treated with a 7 mW (power density of 4 mW/cm2) HeNe (632.8-nm) laser for times ranging from 249 to 2,290 seconds. Graph redrawn from data contained in ().

BIPHASIC LLLT DOSE RESPONSE STUDIES IN CULTURED NEURONS AND TRAUMATIC BRAIN INJURY MODELS IN MICE

LLLT studies on cultured cortical neurons

In order to elucidate the mechanism responsible for the beneficial effect reported by LLLT for brain related disorders, we carried out studies to look into effects of 810 nm laser on different cellular signaling molecules in primary cortical neurons. The primary cortical neurons were isolated from brains taken from embryonic mice. We irradiated the neurons with different fluences of 0.03, 0.3, 3, 10 or 30 J/cm2 delivered at a constant irradiance of 25 mW/cm2, and subsequently the intracellular levels of ROS, mitochondrial membrane potential (MMP) and ATP was measured. The changes in mitochondrial function were studied in terms of ATP and MMP. Low-level light was found to induce a significant increase in ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses but there was a distinctive pattern of a double peak with the first peak coinciding with the other peaks of ATP and MMP at 3 J/cm2 (Figure 8). However in contrast to ATP and MMP there was a second larger rise in ROS at 30 J/cm2 that coincided with the reduction in MMP below baseline. The results of the this study suggested that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. Conversely at higher fluences beneficial mediators are reduced but potentially harmful mediators are increased. Thus this study offered an explanation for the biphasic dose response induced by LLLT.

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Mean expression levels of reactive oxygen species (ROS, measured by MitoSox red fluorescence), mitochondrial membrane potential (MMP, measured by red/green fluorescence ration of JC1 dye) and ATP (measured by firefly luciferase assay) in primary mouse cortical neurons treated with various fluences of 810-laser delivered at 25 mW/cm2 over times varying from 1.2 to 1200 seconds.

LLLT in a mouse model of traumatic brain injury

We have been studying the effect of transcranial laser (810-nm) on mouse models of traumatic brain injury. The model involves a controlled cortical impact using a pneumatic piston device through a craniotomy followed by closure of the head. This injury can be adjusted in severity to produce a neurological severity score (NSS based on a panel of standardized behavioral tests) of 7–8 on a scale of 0 (normal mice) to 10 (severe brain injury that causes death). The basic finding was that delivering a single dose of 36 J/cm2 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head at a time point of 4 hours post-TBI was highly effective in ameliorating the neurological symptoms suffered by the mice (Figure 9A). When we delivered 10 times as much 810-nm laser (360 J/cm2 at 500 mW/cm2) also taking 12 minutes the beneficial effect totally disappeared, and at early time points (1–6 days) the high fluence appeared to be worse than no treatment (Figure 9B).

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Transcranial laser therapy (36 J/cm2 of 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head) was used to treat mice with controlled cortical impact TBI four hours after injury. (A) Significant improvement in neurological severity score continuing for 4 weeks after a single treatment. (B) Delivering ten times more light by increasing irradiance tenfold (500 mW/cm2) loses all therapeutic benefit, and produces worse performance soon after laser. (C) Repeating beneficial laser treatment daily for 14 days loses benefit in performance after 5 days.

When we repeated the effective laser treatments 14 times (36 J/cm2 delivered at 50-mW/cm2 once a day for 14 days starting 4 hours post-TB) we found a very interesting result (Figure 9C). For the first 4 days the improvement in NSS in the repeated laser group was marginally better than the single treatment. However on day 5 the gradual improvement ceased and as the laser was repeated the NSS got closer to that of untreated TBI mice until at day 14 it actually crossed over. Although the differences were not statistically significant it appeared that from day 16 until day 28 the mice that received 14 laser treatments did worse than those that received no treatment at all.

POSSIBLE EXPLANATIONS FOR BIPHASIC DOSE RESPONSE IN LLLT

The triphasic dose response we have observed for ROS production in cultured cortical neurons (see Fig 7) suggests an explanation for the biphasic dose response. The hypothesis is that there are two kinds of ROS. Good ROS are produced at fairly low fluences of light. The reason for the production of good ROS is likely to be connected with stimulation of mitochondrial electron transport as shown by increases in MMP and increases in ATP production. These good ROS can initiate beneficial cell signaling pathwas leading to activation of redox sensitive transcription factors such as NF-κB (; ). NF-κB activation induces expression of a large number of gene products related to cell proliferation and survival (; ). As the fluence of light is increased the beneficial ROS production in the mitochondria decreases in tandem with reductions in MMP and a drop-off in ATP production. Then when even more light is delivered there is a second peak in ROS production, which we will call bad ROS. Bad ROS can damage the mitochondria leading to a drop in MMP below baseline levels and presumably can lead to initiation of apoptosis by the mitochondrial pathway including cytochrome c release. It remains to be seen whether the good and bad ROS are identical species and just differ in amount, or whether they are chemically different species. For instance it may be hypothesized that the good ROS consists mainly of superoxide while the bad ROS consists of more damaging ROS such as hydroxyl radicals and peroxynitrite. In Figure 7 we used just one type of fluorescent ROS indicator (mitoSOX red), which is commonly supposed to be specific for superoxide but will likely also be activated by hydroxyl radicals and peroxynitrite.

There have been several studies showing that relatively high doses of light can induce apoptosis in various cell types via ROS-mediated signaling pathways (). Meanwhile, there is an important proapoptotic signaling pathway has been identified which involv


Original Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315174/

Impact of Photobiomodulation on T3/T4 Ratio and Quality of Life in Hashimoto Thyroiditis

Candas Ercetin , Nuri Alper Sahbaz , Sami Acar , Firat Tutal , and Yesim Erbil - Photobiomodulation, Photomedicine, and Laser Surgery (Publication)
PBM causes major improvements in HT-related symptoms of the patient.
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Objective and background: Hashimoto's thyroiditis (HT) is both a B cell- and T cell-mediated, organ-specific autoimmune disease. No current treatment for underlying pathological mechanisms is available for HT and once diagnosed it requires long-term levothyroxine (LT4) treatment in most patients. The aim of our study was to evaluate the effects of photobiomodulation (PBM) on HT patients regarding thyroid functions, thyroid autoantibody levels, and decrease in hormone replacement needs.
Conclusions: In conclusion, our results are encouraging and PBM seems to be very effective in increasing T3/T4 ratio and decreasing TPO Ab levels and weekly dosages of LT4 replacement therapy. Anti-inflammatory properties of PBM are greatly responsible for these changes and PBM causes major improvements in HT-related symptoms of the patient.

Original Source: https://www.liebertpub.com/doi/10.1089/photob.2019.4740

Low-level laser in the treatment of patients with hypothyroidism induced by chronic autoimmune thyroiditis: a randomized, placebo-controlled clinical trial.

Höfling DB1, Chavantes MC, Juliano AG, Cerri GG, Knobel M, Yoshimura EM, Chammas MC. - Lasers Med Sci. 2013 May;28(3):743-53. doi: 10.1007/s10103-012-1129-9. Epub 2012 Jun 21. (Publication)
These findings suggest that LLLT was effective at improving thyroid function, promoting reduced TPOAb-mediated autoimmunity and increasing thyroid echogenicity in patients with CAT hypothyroidism.
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Intro: Chronic autoimmune thyroiditis (CAT) is the most common cause of acquired hypothyroidism, which requires lifelong levothyroxine replacement therapy. Currently, no effective therapy is available for CAT. Thus, the objective of this study was to evaluate the efficacy of low-level laser therapy (LLLT) in patients with CAT-induced hypothyroidism by testing thyroid function, thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb), and ultrasonographic echogenicity. A randomized, placebo-controlled trial with a 9-month follow-up was conducted from 2006 to 2009. Forty-three patients with a history of levothyroxine therapy for CAT-induced hypothyroidism were randomly assigned to receive either 10 sessions of LLLT (830 nm, output power of 50 mW, and fluence of 707 J/cm(2); L group, n=23) or 10 sessions of a placebo treatment (P group, n=20). The levothyroxine was suspended 30 days after the LLLT or placebo procedures. Thyroid function was estimated by the levothyroxine dose required to achieve normal concentrations of T3, T4, free-T4 (fT4), and thyrotropin after 9 months of postlevothyroxine withdrawal. Autoimmunity was assessed by measuring the TPOAb and TgAb levels. A quantitative computerized echogenicity analysis was performed pre- and 30 days postintervention. The results showed a significant difference in the mean levothyroxine dose required to treat the hypothyroidism between the L group (38.59 ± 20.22 μg/day) and the P group (106.88 ± 22.90 μg/day, P<0.001). Lower TPOAb (P=0.043) and greater echogenicity (P<0.001) were also noted in the L group. No TgAb difference was observed. These findings suggest that LLLT was effective at improving thyroid function, promoting reduced TPOAb-mediated autoimmunity and increasing thyroid echogenicity in patients with CAT hypothyroidism.

Background: Chronic autoimmune thyroiditis (CAT) is the most common cause of acquired hypothyroidism, which requires lifelong levothyroxine replacement therapy. Currently, no effective therapy is available for CAT. Thus, the objective of this study was to evaluate the efficacy of low-level laser therapy (LLLT) in patients with CAT-induced hypothyroidism by testing thyroid function, thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb), and ultrasonographic echogenicity. A randomized, placebo-controlled trial with a 9-month follow-up was conducted from 2006 to 2009. Forty-three patients with a history of levothyroxine therapy for CAT-induced hypothyroidism were randomly assigned to receive either 10 sessions of LLLT (830 nm, output power of 50 mW, and fluence of 707 J/cm(2); L group, n=23) or 10 sessions of a placebo treatment (P group, n=20). The levothyroxine was suspended 30 days after the LLLT or placebo procedures. Thyroid function was estimated by the levothyroxine dose required to achieve normal concentrations of T3, T4, free-T4 (fT4), and thyrotropin after 9 months of postlevothyroxine withdrawal. Autoimmunity was assessed by measuring the TPOAb and TgAb levels. A quantitative computerized echogenicity analysis was performed pre- and 30 days postintervention. The results showed a significant difference in the mean levothyroxine dose required to treat the hypothyroidism between the L group (38.59 ± 20.22 μg/day) and the P group (106.88 ± 22.90 μg/day, P<0.001). Lower TPOAb (P=0.043) and greater echogenicity (P<0.001) were also noted in the L group. No TgAb difference was observed. These findings suggest that LLLT was effective at improving thyroid function, promoting reduced TPOAb-mediated autoimmunity and increasing thyroid echogenicity in patients with CAT hypothyroidism.

Abstract: Abstract Chronic autoimmune thyroiditis (CAT) is the most common cause of acquired hypothyroidism, which requires lifelong levothyroxine replacement therapy. Currently, no effective therapy is available for CAT. Thus, the objective of this study was to evaluate the efficacy of low-level laser therapy (LLLT) in patients with CAT-induced hypothyroidism by testing thyroid function, thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb), and ultrasonographic echogenicity. A randomized, placebo-controlled trial with a 9-month follow-up was conducted from 2006 to 2009. Forty-three patients with a history of levothyroxine therapy for CAT-induced hypothyroidism were randomly assigned to receive either 10 sessions of LLLT (830 nm, output power of 50 mW, and fluence of 707 J/cm(2); L group, n=23) or 10 sessions of a placebo treatment (P group, n=20). The levothyroxine was suspended 30 days after the LLLT or placebo procedures. Thyroid function was estimated by the levothyroxine dose required to achieve normal concentrations of T3, T4, free-T4 (fT4), and thyrotropin after 9 months of postlevothyroxine withdrawal. Autoimmunity was assessed by measuring the TPOAb and TgAb levels. A quantitative computerized echogenicity analysis was performed pre- and 30 days postintervention. The results showed a significant difference in the mean levothyroxine dose required to treat the hypothyroidism between the L group (38.59 ± 20.22 μg/day) and the P group (106.88 ± 22.90 μg/day, P<0.001). Lower TPOAb (P=0.043) and greater echogenicity (P<0.001) were also noted in the L group. No TgAb difference was observed. These findings suggest that LLLT was effective at improving thyroid function, promoting reduced TPOAb-mediated autoimmunity and increasing thyroid echogenicity in patients with CAT hypothyroidism.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/22718472

Effect of diode laser on proliferation and differentiation of PC12 cells.

Saito K1, Hashimoto S, Jung HS, Shimono M, Nakagawa K. - Bull Tokyo Dent Coll. 2011;52(2):95-102. ()
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Intro: This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and β-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Background: This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and β-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Abstract: Abstract This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and β-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/21701122

Lasers, stem cells, and COPD

Feng Lin1†, Steven F Josephs1†, Doru T Alexandrescu2†, Famela Ramos1, Vladimir Bogin3, Vincent Gammill4, Constantin A Dasanu5, Rosalia De Necochea-Campion6, Amit N Patel7, Ewa Carrier6, David R Koos1* - (Publication)
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 Abstract

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue

healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, nonthermal

intervention that has the potential to modulate regenerative processes is worthy of attention when searching

for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a

“photoceutical” for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis,

and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous

stem cells, as well as post-mobilization directing of stem cells will be discussed.

Introduction (Personal Perspective)

We came upon the field of low level laser (LLL) therapy

by accident. One of our advisors read a press release

about a company using this novel technology of specific

light wavelengths to treat stroke. Given the possible role

of stem cells in post-stroke regeneration, we decided to

cautiously investigate. As a background, it should be

said that our scientific team has been focusing on the

area of cord blood banking and manufacturing of disposables

for processing of adipose stem cells for the past 3

years. Our board has been interested in strategically

refocusing the company from services-oriented into a

more research-focused model. An unbiased exploration

into the various degenerative conditions that may be

addressed by our existing know-how led us to explore

the condition of chronic obstructive pulmonary disease

(COPD), an umbrella term covering chronic bronchitis

and emphysema, which is the 4th largest cause of death

in the United States. As a means of increasing our probability

of success in treatment of this condition, the

decision was made to develop an adjuvant therapy that

would augment stem cell activity. The field of LLL therapy

attracted us because it appeared to be relatively

unexplored scientific territory for which large amounts

of clinical experience exist. Unfortunately, it was difficult

to obtain the cohesive “state-of-the-art” description of

the molecular/cellular mechanisms of this therapy in

reviews that we have searched. Therefore we sought in

this mini-review to discuss what we believe to be relevant

to investigators attracted by the concept of “regenerative

photoceuticals”. Before presenting our synthesis

of the field, we will begin by describing our rationale for

approaching COPD with the autologous stem cell based

approaches we are developing.

COPD as an Indication for Stem Cell Therapy

COPD possesses several features making it ideal for

stem cell based interventions: a) the quality of life and

lack of progress demands the ethical exploration of

novel approaches. For example, bone marrow stem cells

have been used in over a thousand cardiac patients with

some indication of efficacy [1,2]. Adipose-based stem

cell therapies have been successfully used in thousands

of race-horses and companion animals without adverse

effects [3], as well as numerous clinical trials are

ongoing and published human data reports no adverse

effects (reviewed in ref [4]). Unfortunately, evaluation of

stem cell therapy in COPD has lagged behind other

areas of regenerative investigation; b) the underlying

cause of COPD appears to be inflammatory and/or

immunologically mediated. The destruction of alveolar

tissue is associated with T cell reactivity [5,6], pathological

pulmonary macrophage activation [7], and auto-antibody

production [8]. Mesenchymal stem cells have been

demonstrated to potently suppress autoreactive T cells

[9,10], inhibit macrophage activation [11], and autoantibody

responses [12]. Additionally, mesenchymal stem

cells can be purified in high concentrations from adipose

stromal vascular tissue together with high concentrations of T regulatory cells [4], which in animal

models are approximately 100 more potent than peripheral

T cells at secreting cytokines therapeutic for COPD

such as IL-10 [13,14]. Additionally, use of adipose

derived cells has yielded promising clinical results in

autoimmune conditions such as multiple sclerosis [4];

and c) Pulmonary stem cells capable of regenerating

damaged parenchymal tissue have been reported [15].

Administration of mesenchymal stem cells into neonatal

oxygen-damaged lungs, which results in COPD-like

alveoli dysplasia, has been demonstrated to yield

improvements in two recent publications [16,17].

Based on the above rationale for stem cell-based

COPD treatments, we began our exploration into this

area by performing several preliminary experiments and

filing patents covering combination uses of stem cells

with various pharmacologically available antiinflammatories,

as well as methods of immune modulation. These

have served as the basis for two of our pipeline candidates,

ENT-111, and ENT-894. As a commerciallyoriented

organization, we needed to develop a therapeutic

candidate that not only has a great potential for efficacy,

but also can be easily implemented as part of the

standard of care. Our search led us to the area of low

level laser (LLL) therapy. From our initial perception as

neophytes to this field, the area of LLL therapy has been

somewhat of a medical mystery. A pubmed search for

“low level laser therapy” yields more than 1700 results,

yet before stumbling across this concept, none of us, or

our advisors, have ever heard of this area of medicine.

On face value, this field appeared to be somewhat of a

panacea: clinical trials claiming efficacy for conditions

ranging from alcoholism [18], to sinusitis [19], to

ischemic heart disease [20]. Further confusing was that

many of the studies used different types of LLL-generating

devices, with different parameters, in different model

systems, making comparison of data almost impossible.

Despite this initial impression, the possibility that a simple,

non-invasive methodology could exist that augments

regenerative potential in a tissue-focused manner

became very enticing to us. Specific uses envisioned, for

which intellectual property was filed included using light

to concentrate stem cells to an area of need, to modulate

effects of stem cells once they are in that specific

area, or even to use light together with other agents to

modulate endogenous stem cells.

The purpose of the current manuscript is to overview

some of the previous work performed in this area that was

of great interest to our ongoing work in regenerative medicine.

We believe that greater integration of the area of

LLL with current advancements in molecular and cellular

biology will accelerate medical progress. Unfortunately, in

our impression to date, this has been a very slow process.

What is Low Level Laser Irradiation?

Lasers (Light amplification by stimulated emission of

radiation) are devices that typically generate electromagnetic

radiation which is relatively uniform in wavelength,

phase, and polarization, originally described by Theodore

Maiman in 1960 in the form of a ruby laser [21]. These

properties have allowed for numerous medical applications

including uses in surgery, activation of photodynamic

agents, and various ablative therapies in cosmetics that are

based on heat/tissue destruction generated by the laser

beam [22-24]. These applications of lasers are considered

“high energy” because of their intensity, which ranges

from about 10-100 Watts. The subject of the current

paper will be another type of laser approach called low

level lasers (LLL) that elicits effects through non-thermal

means. This area of investigation started with the work of

Mester et al who in 1967 reported non-thermal effects of

lasers on mouse hair growth [25]. In a subsequent study

[26], the same group reported acceleration of wound healing

and improvement in regenerative ability of muscle

fibers post wounding using a 1 J/cm2 ruby laser. Since

those early days, numerous in vitro and in vivo studies

have been reported demonstrating a wide variety of therapeutic

effects involving LLL, a selected sample of which

will be discussed below. In order to narrow our focus of

discussion, it is important to first begin by establishing the

current definition of LLL therapy. According to Posten et

al [27], there are several parameters of importance: a)

Power output of laser being 10-3 to 10-1 Watts; b) Wavelength

in the range of 300-10,600 nm; c) Pulse rate from 0,

meaning continuous to 5000 Hertz (cycles per second); d)

intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/

cm2. Most common methods of administering LLL radiation

include lasers such as ruby (694 nm), Ar (488 and 514

nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647

nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm).

Perhaps one of the most distinguishing features of LLL

therapy as compared to other photoceutical modalities is

that effects are mediated not through induction of thermal

effects but rather through a process that is still not clearly

defined called “photobiostimulation”. It appears that this

effect of LLL is not depend on coherence, and therefore

allows for use of non-laser light generating devices such as

inexpensive Light Emitting Diode (LED) technology [28].

To date several mechanisms of biological action have

been proposed, although none are clearly established.

These include augmentation of cellular ATP levels [29],

manipulation of inducible nitric oxide synthase (iNOS)

activity [30,31], suppression of inflammatory cytokines

such as TNF-alpha, IL-1beta, IL-6 and IL-8 [32-36],

upregulation of growth factor production such as PDGF,

IGF-1, NGF and FGF-2 [36-39], alteration of mitochondrial

membrane potential [29,40-42] due to chromophores found in the mitochondrial respiratory

chain [43,44] as reviewed in [45], stimulation of protein

kinase C (PKC) activation [46], manipulation of NF-!B

activation [47], direct bacteriotoxic effect mediated by

induction of reactive oxygen species (ROS) [48], modification

of extracellular matrix components [49], inhibition

of apoptosis [29], stimulation of mast cell

degranulation [50], and upregulation of heat shock proteins

[51]. Unfortunately these effects have been demonstrated

using a variety of LLL devices in noncomparable

models. To add to confusion, dose-dependency

seems to be confined to such a narrow range or

does not seem to exist in that numerous systems therapeutic

effects disappear with increased dose.

In vitro studies of LLL

In areas of potential phenomenology, it is important to

begin by assessing in vitro studies reported in the literature

in which reproducibility can be attained with some

degree of confidence, and mechanistic dissection is simpler

as compared with in vivo systems. In 1983, one of

the first studies to demonstrate in vitro effects of LLL

was published. The investigators used a helium neon

(He-Ne) laser to generate a visible red light at 632.8 nm

for treatment of porcine granulosa cells. The paper

described upregulation of metabolic and hormone-producing

activity of the cells when exposed for 60 seconds

to pulsating low power (2.8 mW) irradiation [52]. The

possibility of modulating biologically-relevant signaling

proteins by LLL was further assessed in a study using an

energy dose of 1.5 J/cm2 in cultured keratinocytes.

Administration of He-Ne laser emitted light resulted in

upregulated gene expression of IL-1 and IL-8 [53]. Production

of various growth factors in vitro suggests the

possibility of enhanced cellular mitogenesis and mobility

as a result of LLL treatment. Using a diode-based

method to generate a similar wavelength to the He-Ne

laser (363 nm), Mvula et al reported in two papers that

irradiation at 5 J/cm2 of adipose derived mesenchymal

stem cells resulted in enhanced proliferation, viability

and expression of the adhesion molecule beta-1 integrin

as compared to control [54,55]. In agreement with possible

regenerative activity based on activation of stem

cells, other studies have used an in vitro injury model to

examine possible therapeutic effects. Migration of fibroblasts

was demonstrated to be enhanced in a “wound

assay” in which cell monolayers are scraped with a pipette

tip and amount of time needed to restore the

monolayer is used as an indicator of “healing”. The cells

exposed to 5 J/cm2 generated by an He-Ne laser

migrated rapidly across the wound margin indicating a

stimulatory or positive influence of phototherapy.

Higher doses (10 and 16 J/cm2) caused a decrease in

cell viability and proliferation with a significant amount

of damage to the cell membrane and DNA [56]. In

order to examine whether LLL may positively affect

healing under non-optimal conditions that mimic clinical

situations treatment of fibroblasts from diabetic animals

was performed. It was demonstrated that with the

He-Ne laser dosage of 5 J/cm2 fibroblasts exhibited an

enhanced migration activity, however at 16 J/cm2 activity

was negated and cellular damage observed [57]. Thus

from these studies it appears that energy doses from 1.5

J/cm2 to 5 J/cm2 are capable of eliciting “biostimulatory

effects” in vitro in the He-Ne-based laser for adherent

cells that may be useful in regeneration such as fibroblasts

and mesenchymal stem cells.

Studies have also been performed in vitro on immunological

cells. High intensity He-Ne irradiation at 28

and 112 J/cm2 of human peripheral blood mononuclear

cells, a heterogeneous population of T cells, B cells, NK

cells, and monocytes has been described to induce chromatin

relaxation and to augment proliferative response

to the T cell mitogen phytohemaglutin [58]. In human

peripheral blood mononuclear cells (PBMC), another

group reported in two papers that interleukin-1 alpha

(IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha),

interleukin-2 (IL-2), and interferon-gamma (IFNgamma)

at a protein and gene level in PBMC was

increased after He-Ne irradiation at 18.9 J/cm2 and

decreased with 37.8 J/cm2 [59,60]. Stimulation of human

PBMC proliferation and murine splenic lymphocytes

was also reported with He-Ne LLL [61,62]. In terms of

innate immune cells, enhanced phagocytic activity of

murine macrophages have been reported with energy

densities ranging from 100 to 600 J/cm2, with an optimal

dose of 200 J/cm2 [63]. Furthermore, LLL has been

demonstrated to augment human monocyte killing

mycobacterial cells at similar densities, providing a functional

correlation [64].

Thus from the selected in vitro studies discussed, it

appears that modulation of proliferation and soluble factor

production by LLL can be reliably reproduced. However

the data may be to some extent contradictory. For

example, the over-arching clinical rationale for use of

LLL in conditions such as sinusitis [65], arthritis [66,67],

or wound healing [68] is that treatment is associated

with anti-inflammatory effects. However the in vitro studies

described above suggested LLL stimulates proinflammatory

agents such as TNF-alpha or IL-1 [59,60].

This suggests the in vivo effects of LLL may be very

complex, which to some extent should not be surprising.

Factors affecting LLL in vivo actions would include

degree of energy penetration through the tissue, the various

absorption ability of cells in the various tissues, and

complex chemical changes that maybe occurring in

paracrine/autocrine manner. Perhaps an analogy to the

possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal

ozonation of blood. This practice is similar to

LLL therapy given that it is used in treatment of conditions

such as atherosclerosis, non-healing ulcers, and

various degenerative conditions, despite no clear

mechanistic understanding [69-71]. In vitro studies have

demonstrated that ozone is a potent oxidant and inducer

of cell apoptosis and inflammatory signaling [72-74].

In contrast, in vivo systemic changes subsequent to

administration of ozone or ozonized blood in animal

models and patients are quite the opposite. Numerous

investigators have published enhanced anti-oxidant

enzyme activity such as elevations in Mg-SOD and glutathione-

peroxidase levels, as well as diminishment of

inflammation-associated pathology [75-78]. Regardless

of the complexity of in vivo situations, the fact that

reproducible, in vitro experiments, demonstrate a biological

effect provided support for us that there is some

basis for LLL and it is not strictly an area of

phenomenology.

Animal Studies with LLL

As early as 1983, Surinchak et al reported in a rat skin

incision healing model that wounds exposed He-Ne

radiation of fluency 2.2 J/cm2 for 3 min twice daily for

14 days demonstrated a 55% increase in breaking

strength over control rats. Interestingly, higher doses

yielded poorer healing [79]. This application of laser

light was performed directly on shaved skin. In a contradictory

experiment, it was reported that rats irradiated

for 12 days with four levels of laser light (0.0, 0.47, 0.93,

and 1.73 J/cm2) a possible strengthening of wounds tension

was observed at the highest levels of irradiation

(1.73 J/cm2), however it did not reach significance when

analyzed by resampling statistics [80]. In another

wound-healing study Ghamsari et al reported accelerated

healing in the cranial surface of teats in dairy cows

by administration of He-Ne irradiation at 3.64 J/cm2

dose of low-level laser, using a helium-neon system with

an output of 8.5 mW, continuous wave [81]. Collagen

fibers in LLL groups were denser, thicker, better

arranged and more continuous with existing collagen

fibers than those in non-LLL groups. The mean tensile

strength was significantly greater in LLL groups than in

non-LLL groups [82]. In the random skin flap model,

the use of He-Ne laser irradiation with 3 J/cm2 energy

density immediately after the surgery and for the four

subsequent days was evaluated in 4 experimental

groups: Group 1 (control) sham irradiation with He-Ne

laser; Group 2 irradiation by punctual contact technique

on the skin flap surface; Group 3 laser irradiation surrounding

the skin flap; and Group 4 laser irradiation

both on the skin flap surface and around it. The percentage

of necrotic area of the four groups was determined

on day 7-post injury. The control group had an average

necrotic area of 48.86%; the group irradiated on the skin

flap surface alone had 38.67%; the group irradiated

around the skin flap had 35.34%; and the group irradiated

one the skin flap surface and around it had

22.61%. All experimental groups reached statistically significant

values when compared to control [83]. Quite

striking results were obtained in an alloxan-induced diabetes

wound healing model in which a circular 4 cm2

excisional wound was created on the dorsum of the diabetic

rats. Treatment with He-Ne irradiation at 4.8 J/

cm2 was performed 5 days a week until the wound

healed completely and compared to sham irradiated animals.

The laser-treated group healed on average by the

18th day whereas, the control group healed on average

by the 59th day [84].

In addition to mechanically-induced wounds, beneficial

effects of LLL have been obtained in burn-wounds

in which deep second-degree burn wounds were

induced in rats and the effects of daily He-Ne irradiation

at 1.2 and 2.4 J/cm2 were assessed in comparison to

0.2% nitrofurazone cream. The number of macrophages

at day 16, and the depth of new epidermis at day 30,

was significantly less in the laser treated groups in comparison

with control and nitrofurazone treated groups.

Additionally, infections with S. epidermidis and S. aureus

were significantly reduced [85].

While numerous studies have examined dermatological

applications of LLL, which may conceptually be

easier to perform due to ability to topically apply light,

extensive investigation has also been made in the area

of orthopedic applications. Healing acceleration has

been observed in regeneration of the rat mid-cortical

diaphysis of the tibiae, which is a model of post-injury

bone healing. A small hole was surgically made with a

dentistry burr in the tibia and the injured area and LLL

was administered over a 7 or 14 day course transcutaneously

starting 24 h from surgery. Incident energy density

dosages of 31.5 and 94.5 J/cm2 were applied during

the period of the tibia wound healing. Increased angiogenesis

was observed after 7 days irradiation at an

energy density of 94.5 J/cm2, but significantly decreased

the number of vessels in the 14-day irradiated tibiae,

independent of the dosage [86]. In an osteoarthritis

model treatment with He-Ne resulted in augmentation

of heat shock proteins and pathohistological improvement

of arthritic cartilage [87]. The possibility that a

type of preconditioning response is occurring, which

would involve induction of genes such as hemoxygenase-

1 [88], remains to be investigated. Effects of LLL

therapy on articular cartilage were confirmed by another

group. The experiment consisted of 42 young Wistar

rats whose hind limbs were operated on in order to

immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/

cm2, 5.8 W/cm2, and sham treatment. After 6 times of

treatment for another 2 weeks significantpreservation of

articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy

was observed [89].

Muscle regeneration by LLL was demonstrated in a rat

model of disuse atrophy in which eight-week-old rats

were subjected to hindlimb suspension for 2 weeks,

after which they were released and recovered. During

the recovery period, rats underwent daily LLL irradiation

(Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to

the right gastrocnemius muscle through the skin. After

2-weeks the number of capillaries and fibroblast growth

factor levels exhibited significant elevation relative to

those of the LLL-untreated muscles. LLL treatment

induced proliferation in satellite cells as detected by

BRdU [90].

Other animal studies of LLL have demonstrated

effects in areas that appear unrelated such as suppression

of snake venom induced muscle death [91],

decreasing histamine-induced vasospasms [92], inhibition

of post-injury restenosis [93], and immune stimulation

by thymic irradiation [94].

Clinical Studies Using LLL

Growth factor secretion by LLL and its apparent regenerative

activities have stimulated studies in radiationinduced

mucositis. A 30 patient randomized trial of carcinoma

patients treated by radiotherapy alone (65 Gy at

a rate of 2 Gy/fraction, 5 fractions per week) without

prior surgery or concomitant chemotherapy suffering

from radiation-induced mucositis was performed using a

He-Ne 60 mW laser. Grade 3 mucositis occured with a

frequency of 35.2% in controls and at 7.6% of treated

patients. Furthermore, a decrease in “severe pain” (grade

3) was observed in that 23.8% in the control group

experienced this level of pain, as compared to 1.9% in

the treatment group [95]. A subsequent study reported

similar effects [96].

Healing ability of lasers was also observed in a study

of patients with gingival flap incisions. Fifty-eight extraction

patients had one of two gingival flap incisions lased

with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing

rates were evaluated clinically and photographically.

Sixty-nine percent of the irradiated incisions healed faster

than the control incisions. No significant difference

in healing was noted when patients were compared by

age, gender, race, and anatomic location of the incision

[97]. Another study evaluating healing effects of LLL in

dental practice examined 48 patients subjected to surgical

removal of their lower third molars. Treated patients

were administered Ga-Al-As diode generated 808 nm at

a dose of 12 J. The study demonstrated that extraoral

LLL is more effective than intraoral LLL, which was

more effective than control for the reduction of postoperative

trismus and swelling after extraction of the

lower third molar [98].

Given the predominance of data supporting fibroblast

proliferative ability and animal wound healing effects of

LLL therapy, a clinical trial was performed on healing of

ulcers. In a double-blinded fashion 23 diabetic leg ulcers

from 14 patients were divided into two groups. Phototherapy

was applied (<1.0 J/cm2) twice per week, using a

Dynatron Solaris 705(R) LED device that concurrently

emits 660 and 890 nm energies. At days 15, 30, 45, 60,

75, and 90 mean ulcer granulation and healing rates

were significantly higher for the treatment group as

compared to control. By day 90, 58.3% of the ulcers in

the LLL treated group were fully healed and 75%

achieved 90-100% healing. In the placebo group only

one ulcer healed fully [68].

As previously mentioned, LLL appears to have some

angiogenic activity. One of the major problems in coronary

artery disease is lack of collateralization. In a 39

patient study advanced CAD, two sessions of irradiation

of low-energy laser light on skin in the chest area from

helium-neon B1 lasers. The time of irradiation was 15

minutes while operations were performed 6 days a week

for one month. Reduction in Canadian Cardiology

Society (CCS) score, increased exercise capacity and

time, less frequent angina symptoms during the treadmill

test, longer distance of 6-minute walk test and a

trend towards less frequent 1 mm ST depression lasting

1 min during Holter recordings was noted after therapy

[99].

Perhaps one of the largest clinical trials with LLL was

the NEST trial performed by Photothera. In this double

blind trial 660 stroke patients were recruited and randomized:

331 received LLL and 327 received sham. No

prespecified test achieved significance, but a post hoc

analysis of patients with a baseline National Institutes of

Health Stroke Scale score of <16 showed a favorable

outcome at 90 days on the primary end point (P <

0.044) [100]. Currently Photothera is in the process of

repeating this trial with modified parameters.

Relevance of LLL to COPD

A therapeutic intervention in COPD would require

addressing the issues of inflammation and regeneration.

Although approaches such as administration of bone marrow

stem cells, or fat derived cellular components have

both regenerative and anti-inflammatory activity in animal

models, the need to enhance their potency for clinical

applications can be seen in the recent Osiris’s COPD trial

interim data which reported no significant improvement

in pulmonary function [101]. Accordingly, we sought to

develop a possible rationale for how LLL may be useful as

an adjunct to autologous stem cell therapy.

Table 1 Examples of LLL Properties Relevant to COPD

COPD

Property

LLL Experiment LLL Details Ref

Inflammation In vivo. Decreased joint inflammation in zymosan-induced

arthritis

Semiconductor laser (685 nm and 830 nm) at (2.5 J/cm2)

In vitro. Suppression of LPS-induced bronchial inflammation and

TNF-alpha.

655 nm at of 2.6 J/cm2

In vivo. Carrageenan-induced pleurisy had decreased leukocyte

infiltration and cytokine (TNF-alpha, IL-6, and MCP)

660 nm at 2.1 J/cm2

In vitro. LPS stimulated Raw 264.7 monocytes had reduced gene

expression of MCP-1, IL-1 and IL-6

780 nm diode laser at 2.2 J/cm2)

In vivo. Suppression of LPS-stimulated neutrophil influx,

myeloperoxidase activity and IL-1beta in bronchoalveolar lavage

fluid.

660 nm diode laser at 7.5 J/cm2

In vitro. Inhibition of TNF-alpha induced IL-1, IL-8 and TNF-alpha

mRNA in human synoviocytes

810 nm (5 J/cm2) suppressed IL-1 and TNF, (25 J/cm2) also

suppressed IL-8

In vivo. Reduction of TNF-alpha in diaphragm muscle after

intravenous LPS injection.

4 sessions in 24 h with diode Ga-AsI-Al laser of 650 nm and

a total dose of 5.2 J/cm2

In vivo. Inhibition of LPS induced peritonitis and neutrophil influx 3 J/cm2 and 7.5 J/cm2

Growth Factor Production

In vivo. Upregulation of TGF-b and PDGF in rat gingiva after

incision.

He-Ne laser (632.8 nm) at a dose of 7.5 J/cm2

In vitro. Osteoblast-like cells were isolated from fetal rat calvariae

had increased IGF-1

Ga-Al-As laser (830 nm) at (3.75 J/cm2).

In vitro. Upregulated production of IGF-1 and FGF-2 in human

gingival fibroblasts.

685 nm, for 140 s, 2 J/cm2

Angiogenesis

In vivo. Increased fiber to capillary ratio in rabbits with ligated

femoral arteries.

Gallium-aluminum-arsenide (Ga-Al-As) diode laser, 904 nm

and power of 10 mW

In vitro. Stimulation of HUVEC proliferation by conditioned media

from LLL-treated T cells

820 nm at 1.2 and 3.6 J/cm2.

In vitro. 7-fold increased production of VEGF by cardiomyocytes,

1.6-fold increase by smooth muscle cells (SMC) and fibroblasts.

Supernatant of SMC had increased HUVEC-stimulating potential.

He:Ne continuous wave laser (632 nm). 0.5 J/cm2 for SMC,

2.1 J/cm2 for fibroblasts and 1.05 J/cm2 for cardiomyocytes.

In vitro. Direct stimulation of HUVEC proliferation 670 nm diode device at 2 and 8 J/cm2

Direct Stem Cell Effects

In vivo. LLL precondition significantly enhanced early cell survival

rate by 2-fold, decreased the apoptotic percentage of implanted

BMSCs in infarcted myocardium and increased the number of

newly formed capillaries.

635 nm at 0.96 J/cm2

In vitro. LLL stimulated MSC proliferation, VEGF and NGF

production, and myogenic differentiation after 5-aza induction.

635 nm diode laser at 0.5 J/cm2 for MSC proliferation, 5 J/

cm2 for VEGF and NGF production and for augmentation of

induced myogenic differentiation

In vitro. Increased proliferation of rat MSC. Red light LED 630 nm at 2 and 4 J/cm(2)

In vitro. Augmented proliferation of bone marrow and cardiac

specific stem cells.

GA-Al-As 810 nm at 1 and 3 J/cm2

In vitro/In vivo. Administration of LLL-treated MSC resulted 53%

reduction in infarct size, 5- and 6.3-fold significant increase in cell

density that positively immunoreacted to BrdU and c-kit,

respectively, and 1.4- and 2-fold higher level of angiogenesis and

vascular endothelial growth factor, respectively, when compared

to non-laser-treated implanted cells

Ga-Al-As laser (810 nm wavelength), 1 J/cm2

In vitro. Enhanced proliferation of adipose derived MSC in

presence of EGF.

636 nm diode, 5 J/cm2

Lin et al. Journal of Translational Medicine 2010, 8:16

http://www.translational-medicine.com/content/8/1/16

Table 1: Examples of LLL Properties Relevant to COPD (Continued)

In vitro. Enhanced proliferation and beta-1 integrin expression of

adipose derived MSC.

635 nm diode laser, at 5 J/cm2

Clinical. 660 stroke patients: 331 received LLL and 327 received

sham. No prespecified test achieved significance, but a post hoc

analysis of patients with a baseline National Institutes of Health

Stroke Scale score of <16 showed a favorable outcome at 90

days on the primary end point (P < 0.044).

808 nm. No density disclosed.

 

Table 1 depicts some of the properties of LLL that provide

a rationale for the combined use with stem cells. One

of the basic properties of LLL seems to be ability to inhibit

inflammation at the level of innate immune activation.

Representative studies showed that LLL was capable of

suppressing inflammatory genes and/or pathology after

administration of lipopolysaccharide (LPS) as a stimulator

of monocytes [102] and bronchial cells [34], in vitro, and

leukocyte infiltration in vivo [103,104]. Inflammation

induced by other stimulators such as zymosan, carrageenan,

and TNF-alpha was also inhibited by LLL

[32,105,106]. Growth factor stimulating activity of LLL

was demonstrated in both in vitro and in vivo experiments

in which augmentation of FGF-2, PDGF and IGF-1 was

observed [36,37,107]. Endogenous production of these

growth factors may be useful in regeneration based on

activation of endogenous pulmonary stem cells [108,109].

Another aspect of LLL activities of relevance is ability to

stimulate angiogenesis. In COPD, the constriction of

blood vessels as a result of poor oxygen uptake is results

in a feedback loop culminating in pulmonary hypertension.

Administration of angiogenic factors has been

demonstrated to be beneficial in several animal models of

pulmonary pathology [110,111]. The ability of LLL to

directly induce proliferation of HUVEC cells [112], as well

as to augment production of angiogenic factors such as

VEGF [113], supports the possibility of creation of an

environment hospitable to neoangiogenesis which is optimal

for stem cell growth. In fact, a study demonstrated in

vivo induction of neocapillary formation subsequent to

LLL administration in a hindlimb ischemia model [114].

The critical importance of angiogenesis in stem cell

mediated regeneration has previously been demonstrated

in the stroke model, where the major therapeutic activity

of exogenous stem cells has been attributed to angiogenic

as opposed to transdifferentiation effects [115].

Direct evidence of LLL stimulating stem cells has been

obtained using mesenchymal stem cells derived both

from the bone marrow and from the adipose tissue

[116,117]. Interestingly in vivo administration of LLL stimulated

MSC has resulted in 50% decrease in cardiac

infarct size [118]. Clinical translation of LLL has been

performed in the area of stroke, in which a 660 patient

trial demonstrated statistically significant effects in post

trial subset analysis [100].

Conclusions

Despite clinical use of LLL for decades, the field is still

in its infancy. As is obvious from the wide variety of

LLL sources, frequencies, and intensities used, no standard

protocols exist. The ability of LLL to induce

growth factor production, inhibition of inflammation,

stimulation of angiogenesis, and direct effects on stem

cells suggests the urgent need for combining this modality

with regenerative medicine, giving birth to the new

field of “regenerative photoceuticals”. Development of a

regenerative treatment for COPD as well as for other

degenerative diseases would be of considerable benefit.

Regarding COPD, such treatment would be life-saving/

life extending for thousands of affected individuals.

Ceasing smoking or not starting to smoke would considerably

impact this disease.

Acknowledgements

The authors thank Victoria Dardov and Matthew Gandjian for critical

discussions and input.

Author details

1Entest BioMedical, San Diego, CA, USA. 2Georgetown Dermatology,

Washington DC, USA. 3Cromos Pharma Services, Longview, WA, USA. 4Center

for the Study of Natural Oncology, Del Mar, CA, USA. 5Department of

Hematology and Medical Oncology, St Francis Hospital and Medical Center,

Hartford, CT, USA. 6Moores Cancer Center, University of California San Diego,

CA, USA. 7Department of Cardiothoracic Surgery, University of Utah, Salt

Lake City, UT, USA.

Authors’ contributions

FL, SFJ, DTA, FR, VB, VG, CAD, RDNC, ANP, EC, DRK contributed to literature

review, analysis and discussion, synthesis of concepts, writing of the

manuscript and proof-reading of the final draft.

Competing interests

David R Koos is a shareholder, as well as Chairman and CEO of Entest Bio.

Feng Lin is research director of Entest Bio. All other authors declare no

competing interest.

Received: 7 January 2010

Accepted: 16 February 2010 Published: 16 February 2010

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Fisher M, Hacke W, Holt W, Ilic S, Kasner S, Lew R, Nash M, Perez J,

Rymer M, Schellinger P, Schneider D, Schwab S, Veltkamp R, Walker M,

Streeter J, NeuroThera Effectiveness and Safety Trial-2 Investigators:

Effectiveness and safety of transcranial laser therapy for acute ischemic

stroke. Stroke 2009, 40:1359-1364.

101. Osiris Therapeutics Reports Interim Data for COPD Trial. http://www.

medicalnewstoday.com/articles/155267.php.

102. Gavish L, Perez LS, Reissman P, Gertz SD: Irradiation with 780 nm diode

laser attenuates inflammatory cytokines but upregulates nitric oxide in

lipopolysaccharide-stimulated macrophages: implications for the

prevention of aneurysm progression. Lasers Surg Med 2008, 40:371-378.

103. Correa F, Lopes Martins RA, Correa JC, Iversen VV, Joenson J, Bjordal JM:

Low-level laser therapy (GaAs lambda = 904 nm) reduces inflammatory

cell migration in mice with lipopolysaccharide-induced peritonitis.

Photomed Laser Surg 2007, 25:245-249.

104. Aimbire F, Lopes-Martins RA, Castro-Faria-Neto HC, Albertini R,

Chavantes MC, Pacheco MT, Leonardo PS, Iversen VV, Bjordal JM: Low-level

laser therapy can reduce lipopolysaccharide-induced contractile force

dysfunction and TNF-alpha levels in rat diaphragm muscle. Lasers Med

Sci 2006, 21:238-244.

105. de Morais NC, Barbosa AM, Vale ML, Villaverde AB, de Lima CJ, Cogo JC,

Zamuner SR: Anti-Inflammatory Effect of Low-Level Laser and Light-

Emitting Diode in Zymosan-Induced Arthritis. Photomed Laser Surg 2009.

106. Boschi ES, Leite CE, Saciura VC, Caberlon E, Lunardelli A, Bitencourt S,

Melo DA, Oliveira JR: Anti-Inflammatory effects of low-level laser therapy

(660 nm) in the early phase in carrageenan-induced pleurisy in rat.

Lasers Surg Med 2008, 40:500-508.

107. Shimizu N, Mayahara K, Kiyosaki T, Yamaguchi A, Ozawa Y, Abiko Y: Lowintensity

laser irradiation stimulates bone nodule formation via insulinlike

growth factor-I expression in rat calvarial cells. Lasers Surg Med 2007,

39:551-559.

108. Hackett TL, Shaheen F, Johnson A, Wadsworth S, Pechkovsky DV,

Jacoby DB, Kicic A, Stick SM, Knight DA: Characterization of side

population cells from human airway epithelium. Stem Cells 2008,

26:2576-2585.

109. Irwin D, Helm K, Campbell N, Imamura M, Fagan K, Harral J, Carr M,

Young KA, Klemm D, Gebb S, Dempsey EC, West J, Majka S: Neonatal lung

side population cells demonstrate endothelial potential and are altered

in response to hyperoxia-induced lung simplification. Am J Physiol Lung

Cell Mol Physiol 2007, 293:L941-951.

110. Thebaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F,

Hashimoto K, Harry G, Haromy A, Korbutt G, Archer SL: Vascular

endothelial growth factor gene therapy increases survival, promotes

lung angiogenesis, and prevents alveolar damage in hyperoxia-induced

lung injury: evidence that angiogenesis participates in alveolarization.

Circulation 2005, 112:2477-2486.

111. Thebaud B: Angiogenesis in lung development, injury and repair:

implications for chronic lung disease of prematurity. Neonatology 2007,

91:291-297.

112. Schindl A, Merwald H, Schindl L, Kaun C, Wojta J: Direct stimulatory effect

of low-intensity 670 nm laser irradiation on human endothelial cell

proliferation. Br J Dermatol 2003, 148:334-336.

113. Kipshidze N, Nikolaychik V, Keelan MH, Shankar LR, Khanna A, Kornowski R,

Leon M, Moses J: Low-power helium: neon laser irradiation enhances

production of vascular endothelial growth factor and promotes growth

of endothelial cells in vitro. Lasers Surg Med 2001, 28:355-364.

114. Ihsan FR: Low-level laser therapy accelerates collateral circulation and

enhances microcirculation. Photomed Laser Surg 2005, 23:289-294.

115. Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H,

Tsukamoto Y, Iso H, Fujimori Y, Stern DM, Naritomi H, Matsuyama T:

Administration of CD34+ cells after stroke enhances neurogenesis via

angiogenesis in a mouse model. J Clin Invest 2004, 114:330-338.

116. Li WT, Leu YC: Effects of low level red-light irradiation on the

proliferation of mesenchymal stem cells derived from rat bone marrow.

Conf Proc IEEE Eng Med Biol Soc 2007, 2007:5830-5833.

117. Tuby H, Maltz L, Oron U: Low-level laser irradiation (LLLI) promotes

proliferation of mesenchymal and cardiac stem cells in culture. Lasers

Surg Med 2007, 39:373-378.

118. Tuby H, Maltz L, Oron U: Implantation of low-level laser irradiated

mesenchymal stem cells into the infarcted rat heart is associated with

reduction in infarct size and enhanced angiogenesis. Photomed Laser

Surg 2009, 27:227-233.

119. Agaiby AD, Ghali LR, Wilson R, Dyson M: Laser modulation of angiogenic

factor production by T-lymphocytes. Lasers Surg Med 2000, 26:357-363.

120. Zhang H, Hou JF, Shen Y, Wang W, Wei YJ, Hu S: Low Level Laser

Irradiation Precondition to Create Friendly Milieu of Infarcted

Myocardium and Enhance Early Survival of Transplanted Bone Marrow

Cells. J Cell Mol Med 2009.

121. Hou JF, Zhang H, Yuan X, Li J, Wei YJ, Hu SS: In vitro effects of low-level

laser irradiation for bone marrow mesenchymal stem cells: proliferation,

growth factors secretion and myogenic differentiation. Lasers Surg Med

2008, 40:726-733.

doi:10.1186/1479-5876-8-16

Cite this article as: Lin et al.: Lasers, stem cells, and COPD. Journal of

Translational Medicine 2010 8:16.


Original Source: http://www.translational-medicine.com/content/8/1/16

Home Search Introduction

Ken Teegardin - (Video)
View Resource

Welcome to the laser-therapy.us research tool. This tool is a searchable collection of technical publications, books, videos and other resources about the use of lasers for photobiomodulation. This tool includes almost the entire U.S. library of medicine research papers on LLLT, videos from Youtube associated with therapy lasers and the tables of contents from laser therapy books. This allows users to search for a keyword or condition and see resources about using lasers to treat that condition. All the resources include links to the original source so we are not making any statement about the use of lasers for treating non-FDA cleared application, we are simple summarizing what others have said.  Where every possible, we have included a link to the orginal publication.

Here are some of our favorite queries:

This tool uses a broad match query so:

  • It does not correct spelling and searches only cold laser related subjects so do not use LLLT, cold or laser in the search bar
  • It works better with shorter search terms or even parts of search terms
  • It searches all the available fields so you can enter a body part, author, condition or laser brand.
  • Where ever possible, the detailed section about the resource will link to the sources.
  • This system is only for photobiomodulation or cold laser therapy research (including LLLT, laser acupuncture and high power laser therapy) only. It does NOT include photodynamic laser therapy (where the laser is used to react with a pharmaceutical), hot surgery lasers or cosmetic lasers. It does include some resources on weight loss and smoking cessation.

The results of the search are sorted based on 3 quality factors on a scale of 1 to 10 with 10 being the best score. Originally all the resources were given a 5-5-5 until they could be individually evaluated. These scores are purely opinion and are only used to simplify the rank of the results from more valuable to least valuable. This should not be considered a critique of any work. This system was created to help researchers (including ourselves) find the most usable resources for any cold laser therapy research. The resources are assigned values based on the following 3 factors:

  • Efficacy: The resource (especially research papers) should show a significant improvement in the condition being treated. Resources that show better results are given a higher quality score.
  • Detail: The source must give enough information that the results can be duplicated. If a resource lacks too many details that it cannot be recreated, it is given a lower detail score.
  • Lack of Bias: Many resources are created to try and show that one device is superior to its competition. Many manufacturers have staff that crank out biased papers on a regular basis on the hope that this will make their product look superior. If the author of the resource is paid by a manufacturer of the resource appears to be biased towards one device and not one technology, the resource has much less value.

Over the past few years of working with research, we found that a majority of the published resources are lacking in one of these three ranking factors.
The original goal of this research tool was to tie published resources to the protocols in the laser-therapy.us library. This connection allows users to trace each protocol back to a list of resources so the protocol can be researched and improved.

General Comments


POWER
When many of the first research papers were published, the most power laser available for therapy were less than 100mW and many systems had to be pulsed to keep the laser from burning out too quickly. Today, system are available that will deliver up to 60,000mW of continuous output. Because of these power limitation, many early studies were limited to extremely low dosages by today’s standards. It takes a 50mW system 17 minutes to deliver 50 joules at the surface of the skin. If this was spread over a large area of damage or was treating a deeper problem, the actual dosages were much less than 1J/cm2.  Today, we know that these dosages typically produce very little or no results.
WAVELENGTH
About 80% of the resources in this database are in the near infrared wavelength. There is also some interest in the red wavelength (600 to 660nm) . Other wavelengths like blue, purple, and green have very little scientific research behind them and have not gotten much traction in the core therapy market with the exception of some fringe consumer products.
Legal Disclaimer
This research tool is free to use but we make no claims about the accuracy of the information. It is an aggregation of existing published resources and it is up to the user to determine if the source of the resources has any value. The information provided through this web site should not be used for diagnosing or treating a health problem or disease. If you have or suspect you may have a health problem, you should consult your local health care provider.



The query result(s) can be shared using the following direct link. Anyone who clicks on this link in an email or on a web site will be shown the current results for the query.
https://www.laser-therapy.us/research/index.cfm?researchinput=Hashimoto