# Lasers, laser needles, pulsed electromagnetic fields and light emitting diodes are the right tools for healing today’s complex patients and for your practice success!

All devices pictured above and more will likely be available for you to train and practice with in this course.

David Rindge and Healing Light Seminars have been teaching and practicing with energy-based therapies since 2002. We continually update our methods and equipment as new technology and information become available.  We will only offer a device if we are continuing to use it clinically, have found it effective and to deliver good value.

Day 1 focuses on theory, biological effects and essentials for treatment success.   You have the opportunity for hands-on practice with state-of-the-art laser, laser needle acupuncture, pulsed electromagnetic field and light emitting diode therapy systems  systems for the treatment of pain, head to toe.

In Day 2, you will learn how laser, laser needle, light emitting diode and pulsed electromagnetic field therapy devices may be applied successfully in aesthetics / dermatology / facial rejuvenation, cardiovascular disease, digestive, ear and eye disorders, gynecology, for hair regrowth, neuropathy, osteoporosis, respiratory disorders, sports medicine and much more.

This popular training manual by Blahnik and Rindge presents the theory and clinical application of laser therapy in clearly understandable terms with treatment protocols for more than 40 conditions.  Laser Therapy: A Clinical Manual is an important important resource in the course and a $79.00 value. You will also receive treatment protocols for other conditions, updates and much, much more relevant material in this course. Gain a solid understanding of the principles, technology and parameters of energy-based therapies and the skills to apply them successfully in your practice! Our goal is to provide you with everything you need to come from knowledge and strength in your practice with laser, laser needle, pulsed electromagnetic field and light emitting diode therapies. Learn More and Register Here. # Course Date / Location ### November 4-5, 2017. Wild Manta, 5151 South Babcock St, Palm Bay, FL 32905. (321) 676-8606. ## LEARN MORE AND REGISTER HERE ## Or call 321-751-7001. ## Healing Light Seminars Training in Energy-based Therapies since 2002 14 PDAs – NCCAOM 322-5 14 CEUs Florida Acupuncturists ## Think Outside the Box! How does it feel to see immediate and significant improvements for most, perhaps nearly all, patients? Is there a worthwhile specialty you could provide or a group of patients you especially enjoy working with? Energy-based therapies are the right tools help your patients and to increase the possibilities in your practice and improve your bottom line! Gain a solid understanding of the principles, technology and skills for success with energy-based therapies. Our goal is to provide you with everything you need to come from knowledge and strength in your practice with lasers, laser needle acupuncture, light emitting diode and pulsed electromagnetic field therapies. Learn More and Register Here. # Course Date / Location November 4-5, 2017. Wild Manta, 5151 South Babcock St, Palm Bay, FL 32905. (321) 676-8606. ## LEARN MORE AND REGISTER HERE ## Or call 321-751-7001. ## Healing Light Seminars Training in Energy-based Therapies since 2002 14 PDAs – NCCAOM 322-5 14 CEUs Florida Acupuncturists ## Inspire and Deepen Your Practice! # Laser, laser needle acupuncture, pulsed electromagnetic field and led therapies are the right tools for healing today’s complex patients and for your practice success! All devices pictured above and more will likely be available for you to train and practice with in this course. David Rindge and Healing Light Seminars have been teaching and practicing with energy-based therapies since 2002. We continually update our methods and equipment as new technology and information become available. We will only offer a device if we are continuing to use it clinically, have found it effective and to deliver good value. Day 1 focuses on theory, biological effects and essentials for treatment success. You have the opportunity for hands-on practice with state-of-the-art laser, laser needle acupuncture, pulsed electromagnetic field and light emitting diode therapy systems for the treatment of pain, head to toe. In Day 2, you will learn how laser, laser needle, light emitting diode and pulsed electromagnetic field therapy devices may be applied successfully in aesthetics / dermatology / facial rejuvenation, cardiovascular disease, digestive, ear and eye disorders, gynecology, for hair regrowth, neuropathy, osteoporosis, respiratory disorders, sports medicine and much more. ### You will receive Laser Therapy: A Clinical Manualas part of the course. This popular training manual by Blahnik and Rindge presents the theory and clinical application of laser therapy in clearly understandable terms with treatment protocols for more than 40 conditions. Laser Therapy: A Clinical Manual is an important important resource in the course and a$79.00 value.  You will also receive treatment protocols for other conditions, updates and much, much more relevant material in this course.

Gain a solid understanding of the principles, technology and parameters of energy-based therapies and the skills to apply them successfully in your practice!  Our goal is to provide you with everything you need to come from knowledge and strength in your practice with laser, laser needle, pulsed electromagnetic field and light emitting diode therapies.  Learn More and Register Here.

# Course Date / Location

November 4-5, 2017.  Wild Manta, 5151 South Babcock St, Palm Bay, FL 32905.  (321) 676-8606.

## Healing Light Seminars

Training in Energy-based Therapies since 2002

14 PDAs – NCCAOM 322-5

14 CEUs Florida Acupuncturists

## The Promise of Energy-Based Therapies in Pain

by David Rindge.

Acute pain inadequately treated frequently becomes chronic.  Chronic pain is the leading cause of disability.  We spent $2.6 trillion on health care in the U.S. in 2010, 75% of it ($1.95 trillion) to “manage” chronic inflammatory conditions.  The price is too high and consumer satisfaction too low to continue as we have been.

A growing body of science and clinical experience has shown that energy-based treatments can improve outcomes in chronic pain and also suggests health care costs will be reduced.   Low intensity lasers, leds and pulsed electromagnetic fields have well documented properties to move the body through inflammation and heal injury.  Applied at the outset, these methods hold promise to prevent many, perhaps most, acute conditions from ever becoming chronic. What would be the potential health and financial benefits for all Americans – and the economy – were these treatments to be broadly implemented and reimbursable by insurance?

“Arthritis or rheumatism” is the #1 cause of disability = 8.6 million people.
“Back or Spine Problems” is the #2 cause of disability = 7.6 million people
16.2 million people

To see what researchers have reported about the effects of low intensity laser, led and pulsed electromagnetic field therapies to improve the underlying pathology in the #1 and #2 causes of disability, click on:

Chronic Pain is the Leading Cause of Disability

Chronic pain affects more than one out of three U.S. citizens (>100 million). Direct costs have been estimated at $100 billion[1]. Yet though over the counter and prescription pain relievers are expensive, these costs, missed work days and the like are trivial compared to the hidden financial burden, pain and suffering born by individuals and society. Taking care of the disabled is enormously expensive. How expensive? Nobody seems to know exactly. Besides their enormous cost, current methods may do little to address the underlying causes. How much has the high cost of current health care methods contributed to America’s economic tailspin. Can we do better? The best possible treatments for pain would address underlying causes, be safe and affordable. Low intensity laser, led and pulsed electromagnetic field therapies have been documented to heal tissue, promote healthy function and to improve the underlying pathology in the #1 and #2 causes of disability. Therapies documented to heal damaged tissue and improve physiological function are also likely to have an endpoint. As such, they hold great promise to improve the way we care for one another – while also lowering costs. In contrast, “managing” chronic pain endlessly with prescription or over the counter medications is hugely expensive and may do little to address the underlying disease process or to improve health. In 2010 the Patient Protection and Affordable Health Care Act (aka Obamacare) became law. Since 2014 Americans have been required to buy insurance. Does yours include laser, led and pulsed electromagnetic field therapies? Energy-Based Treatments Promise to Lower Costs and Improve Quality of Care As we spent$2.6 trillion on health care in 2010, an average of of 8,402 per person, redistributing expensive methods which have created a seemingly impossible financial burden seems unlikely to this writer to achieve the savings necessary to make the Affordable Care Act viable. By opening to innovation, we can raise the bar in health care, improving quality and making it affordable. Low level lasers, leds and pulsed electromagnetic fields have properties shown to move the body through inflammation to heal damaged tissue, preventing chronic illness. Even in long-standing disease, these methods can restore function, reduce pain and improve quality of life. How much pain, suffering and money will be spared with the implementation of these methods as first line treatments for pain in health care? What might it mean for the health of all Americans and the economy? [1] Bjordal JM, Couppe C, Chow RT, Tuner J, Ljunggren EA, A Systematic Review of Low Level Laser Therapy with Location-Specific Doses for Pain from Chronic Joint Disorders, J Physiother. 2003;49(2):107-16. [i] From http://www.allbusiness.com/labor-employment/compensation-benefits-wages-salaries/12503910-1.html, True cost of disability is staggering.Colorado Springs Business Journal. June 5, 2009 Copyright 2013-2016 by David Rindge. All rights reserved. ## How Laser Therapy Works Laser therapy energizes living systems. Four well documented effects in the scientific literature are: 1. Biostimulation / Tissue Regeneration 2. Reduction of Inflammation 3. Analgesia 4. Enhanced Immune Function / Antimicrobial The most important way in which laser therapy adds energy is through photon absorption by mitochondria. These tiny organelles which have been called the “powerhouses” of the cell, are found in most plants and animals. Mitochondria are able to absorb laser light which then activates a series of reactions to increase and store more cellular energy in the form of adenosine triphosphate (ATP). By increasing energy available in this readily accessible form, laser light is able to greatly stimulate the biological function of cells, tissue, and systems and even raise overall vital energy throughout the individual. “Bio” stimulation = Life stimulation! ### When energy is available, the body can heal itself. Laser therapy has been shown to stimulate the regeneration of bone, blood, the lining of blood vessels, cartilage, nerve, muscle and much more. Moreover, it has been documented to enhance the quality of healed tissue. Laser therapy may be an ideal treatment. It may not only effectively address many medical conditions but also has been widely reported to improve health and wellbeing as evidenced by a host of biological markers. ## Melbourne and Palm Bay, Florida Hotels Our November 2017 seminar will be presented at: 5151 South Babcock Street Palm Bay, Florida 32905 The list of hotels below is organized from closest to farthest from Wild Manta. Holiday Inn Express Palm Bay, 1206 Malabar Rd SE Palm Bay, FL 32907, 321-220-2003 1.9 miles. Residence Inn Melbourne, 1430 S Babcock St, Melbourne, FL 32901, (321) 723-5740 4.3 miles. Hilton Melbourne Rialto Place, 200 Rialto Place, Melbourne, FL 32901, (321) 768-0200 4.8 miles. Courtyard by Marriott Melbourne West, 2101 W New Haven Ave, Melbourne, FL 32901, (321) 724-6400. 5.7 miles Fairfield Inn & Suites, 4355 New Haven Ave, Melbourne, FL 32904, (321) 722-22208.3 miles. Melbourne All Suites Inn, 4455 New Haven Ave, Melbourne, FL 32904, (321) 724-5840, 8.4 miles. If there is anything we can do to help make your educational or travel experience better, please contact us by phone at or by email at info@cooperativemedicine.com. Thank you for your interest and participation! ## Glioblastoma Int J Environ Res Public Health. 2016 Nov; 13(11): 1128. Published online 2016 Nov 12. doi: 10.3390/ijerph13111128 PMCID: PMC5129338 # An Overview of Sub-Cellular Mechanisms Involved in the Action of TTFields Jack A. Tuszynski,1,2,* Cornelia Wenger,3 Douglas E. Friesen,1 and Jordane Preto1 Mats-Olof Mattsson, Academic Editor 1Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada; ac.atreblau@neseirfed (D.E.F.); moc.liamg@oterp.enadroj (J.P.) 2Department of Physics, University of Alberta, Edmonton, AB T6G 2E1, Canada 3The Institute of Biophysics and Biomedical Engineering, Faculdade de Ciências, Universidade de Lisboa, Lisboa 1749-016, Portugal; tp.lu.cf@regnewc *Correspondence: ac.atreblau@tkcaj; Tel.: +1-780-432-8906 Received 2016 Jun 26; Accepted 2016 Nov 7. ## Abstract Long-standing research on electric and electromagnetic field interactions with biological cells and their subcellular structures has mainly focused on the low- and high-frequency regimes. Biological effects at intermediate frequencies between 100 and 300 kHz have been recently discovered and applied to cancer cells as a therapeutic modality called Tumor Treating Fields (TTFields). TTFields are clinically applied to disrupt cell division, primarily for the treatment of glioblastoma multiforme (GBM). In this review, we provide an assessment of possible physical interactions between 100 kHz range alternating electric fields and biological cells in general and their nano-scale subcellular structures in particular. This is intended to mechanistically elucidate the observed strong disruptive effects in cancer cells. Computational models of isolated cells subject to TTFields predict that for intermediate frequencies the intracellular electric field strength significantly increases and that peak dielectrophoretic forces develop in dividing cells. These findings are in agreement with in vitro observations of TTFields’ disruptive effects on cellular function. We conclude that the most likely candidates to provide a quantitative explanation of these effects are ionic condensation waves around microtubules as well as dielectrophoretic effects on the dipole moments of microtubules. A less likely possibility is the involvement of actin filaments or ion channels. Keywords: electric fields, biological cells, cancer cells, microtubules, ions, TTFields ## 1. Introduction The effects of external electric fields on biological cells have been extensively studied both in the direct current (DC) and alternating current (AC) cases []. In order to elucidate possible impact of electric fields on cells, various experimental assays as well as analytical and computational models have been developed in the past. Experimentally obtained findings were further translated into biomedical applications. While DC or low-frequency AC fields are used to induce stimulation of excitable cells through membrane depolarization or to promote wound healing, high-frequency AC fields are associated with tissue heating and membrane rupture, thus finding its application in diathermy or ablation techniques. Intermediate-frequency AC electric fields in the kHz to MHz range were commonly assumed to lead to no significant biological effects []. However, in a major breakthrough paper, Kirson et al. [] reported the discovery that low-intensity (1–3 V/cm), intermediate frequency (100–300 kHz) electric fields have a profoundly inhibitory effect on the growth rate of various mammalian tumor cell lines [,,]. This discovery has been translated into a clinical application termed Tumor Treating Fields (TTFields). Based on the results of a Phase III clinical trial [], TTFields have been approved by the United States Food and Drug Administration (FDA) in 2011 for the treatment of recurrent glioblastoma multiforme (GBM) and their efficacy in treating other solid tumor types is currently being investigated clinically []. In late 2015, TTFields were also approved for newly diagnosed GBM patients in combination therapy with temozolomide [,] due to significantly increased survival times. It should be noted that electromagnetic (EM) fields may affect the regulation of cellular growth and differentiation, including the growth of tumors [,]. Both static magnetic and electric fields have altered the mitotic index and cell cycle progression of a number of cell types in various species []. EM low-frequency fields in the range of 50–75 Hz cause perturbations in the mitotic activity of plant and animal cells and a significant inhibitory effect on mitotic activity occurs early during exposure [,,]. While the field amplitudes used are consistent with those of interest to this report, the frequencies are orders of magnitude lower. The reduction in the cell number due to an application of TTFields was studied by in vitro experiments with various cancer cell lines. A significant prolongation of mitosis was predicted, where treated cells remain stationary at metaphase for several hours, which was accompanied by abnormal mitotic figures as well as membrane rupture and blebbing leading to apoptosis [,]. Furthermore, these experiments showed that the inhibitory effect increases with an increasing electric field intensity, resulting in a complete proliferation arrest of rat glioma cells after 24 h exposure to a field intensity of 2.25 V/cm. Additionally, the effects of TTFields have been shown to be frequency-dependent, with a cancer cell line-specific peak frequency of the maximal inhibitory effect, e.g., 200 kHz for glioma cells []. Following these experimental results, two specific mechanisms of action of TTFields have been proposed [,,] which we describe below. Firstly, the applied field is expected to interfere with proper microtubule (MT) formation preventing a functioning mitotic spindle, due to the force of interactions with the large intrinsic dipole moments of the tubulin dimers [,,] that make up MTs. It has been hypothesized that the tubulin dimers might align parallel to the direction of the applied electric field, rather than along the MT axis. Secondly, the cellular morphology during cytokinesis gives rise to a non-uniform intracellular electric field, with a high density at the cleavage furrow between the dividing cells. This non-uniform field leads to the development of dielectrophoretic (DEP) forces [] acting on polarizable macromolecules such as MTs, organelles and all charged structures present in the cell, such as ions, proteins or DNA. Thus, TTFields are considered to be suitable as a novel anti-mitotic cancer treatment modality. In fact, it has been suggested by numerous researchers that endogenous electric fields may play a key role during mitosis. Similar to Cooper [], Pohl et al. [] proposed that the onset of mitosis is associated with a ferroelectric phase transition, which establishes an axis of oscillation for the cellular polarization wave. The mitotic spindle apparatus would delineate the polarization field with MTs lined up along the electric field lines. The poles are expected to experience the highest field intensities while the equatorial plane is likely to provide a nodal manifold for the fields. Consequently, the chromosome condensation during this transformation was predicted to be induced by the static dielectric polarization of the chromatin complex as a result of the cellular ferroelectric phase transition. These conclusions have been supported by experimental evidence for peak EM activity during mitosis [,] and by physical modeling of the electrostatic forces generated by MTs which generate mechanical force required for chromosome segregation during mitosis and influence chromosomal motion [,,]. A detailed review of this aspect can be found elsewhere []. Put together, there is reasonable evidence that especially during mitosis, electric field effects are relevant for the functioning of a dividing cell, especially in the creation of the mitotic spindle. However, to date a rigorous quantitative analysis of the magnitude of these effects within cells exposed to TTFields has not been performed. Furthermore, an analysis of how TTFields might interact with subcellular structures has also never been reported. In a quantitative model, which attempts to explain these effects, an energetic constraint, both from below and above, must be kept in mind. Firstly, for an effect to be of significance at a molecular level, its interaction energy must exceed thermal energy, i.e., kT per degree of freedom (i.e., 4 × 10?21 J). Otherwise, thermal fluctuations will disrupt the action of electric fields. Secondly, it must not produce so much thermal energy as to seriously increase the temperature of the cell. In terms of practical comparisons, a cell generates approximately 3 × 10?12 W of power (3 × 10?12 J/s), much of which is used to maintain a constant physiological temperature. This is found from a simple estimate of energy production by the human body which is 100 W divided by the number of cells in the body which is approximately 3 × 1013 []. In terms of subcellular forces at work, a minimal amount of useful force at a nanometer scale is 1 pN. Motor proteins generate forces on the order of several pN. A force of 1 pN applied to a tip of a microtubule may be used to bend it by as much as 1 µm []. Below, we review electric conduction effects for subcellular structures of interest. The paper is structured as follows. In Section 2, we review what is known about the shape and intensity of the electric field within cells exposed to externally applied electric fields, focusing on cells during mitosis. As a preparation for following sections, Section 3 offers a general introduction to subcellular electrical conduction and electrostatics. Section 4 and Section 5 are devoted to a comprehensive review of the literature concerned with the effects of electric fields on biopolymers, and with the identification of additional mechanisms by which TTFields might interact with cells. Section 4 covers electric field interactions with the cell membrane and the cytosol, whereas the focus of Section 5 penetrates deeper into the cell, shedding light on the electric field effects on subcellular structures of interest, i.e., microtubules (MTs), actin filaments (AFs), ionic charges and DNA. Finally, in Section 6, we present a discussion about the significance of our findings and about future directions of research that should be undertaken in this area. We hope this paper will set a solid theoretical foundation for future studies into the biophysics of TTFields. 2. Induced Electric Fields within Biological Cells in Mitosis The topic of induced electric fields in and around biological cells subject to DC or AC fields has been investigated for decades. The preliminary and most popular studies on the analytical description of steady-state trans-membrane potential induced on spherical cells go back to the work of H.P. Schwan and colleagues []. Arguments were presented to account for the influence of the membrane conductance, surface admittance and spatial charge effects [], as well as for the geometric and material properties of the cell and the surrounding medium []. The impact of external electric fields on a living cell significantly depends on the cell’s shape. Concerning analytical solutions for non-spherical cell shapes, many authors proposed appropriate adaption of the governing equations going back to the work presented in Reference []. Later models aimed to study electric polarization effects on oblate and prolate homogeneous and single-shell spheroids have been developed []. They were later extended to arbitrarily oriented cells of the general ellipsoidal shape []. Importantly, the induced electric field inside a spherical cell is uniform, whereas increasing non-uniformities are predicted for deviations of the regular shape. Another important aspect is the frequency-dependency of the induced trans-membrane voltage and thus also the intracellular field strength, as predicted by the above-mentioned studies and additional research reported elsewhere [,,,,]. For low frequencies, the intracellular space is shielded to a large degree from extracellular electric fields. For example, the electric field strength inside a typical spherical cell is approximately five orders of magnitude lower than that outside the cell [,]. However, as the frequency of the field increases, the high membrane field gain diminishes, allowing for higher field intensities to penetrate into the cell. Recently, Wenger et al. [] developed a computational model to study the application of TTFields to isolated cells during mitosis, specifically during metaphase and at different stages of cytokinesis. Comsol Multiphysics (www.comsol.com) was used to solve for the scalar electric potential V for frequency ranges between 60 Hz and 10 GHz. With voltages of opposite signs set as boundary conditions, a uniform field of 1 V/cm was induced in the model domain. Following 3D confocal microscopy findings [,], the metaphase cell was represented by a sphere with a 10 µm radius and three different stages of cytokinesis were modeled with increased distance between the elliptical mother and daughter cell (see Figure 1, left panel). Three model domains, the extracellular space, the cytoplasm and the membrane, were assigned typical dielectric properties, electrical conductivity and relative permittivity []. (Left) Schematic diagrams of the cell geometries for metaphase and three stages of cytokinesis. Black lines indicate the electric field contours. (Right) The maximum intracellular electric field strength in V/cm plotted as a function of field frequency For a spherical cell during metaphase, the modeling results predict that for frequencies lower than 10 kHz only small changes of the field are detected and the intracellular field strength, Ei, almost equals zero. A first significant increase of Ei is observed at approximately 200 kHz, and Ei increases rapidly as the frequencies increase above this value. This can be seen in the inset of the right panel in Figure 1, which shows a zoomed view of the blue M-phase cell. This transition region depends on the dielectric properties of the cell and its membrane. Nonetheless, above 1 MHz electric current is shunted across the membrane and the impedance is dominated by the cytoplasm. Thus, for an increasing frequency, the electric field inside the cell is augmented and at 1 GHz the cellular structure becomes ‘‘electrically invisible’’ as previously reported []. The directions of the electrical field near the cell membrane resemble already predicted results []. The model further showed that within the dividing cell the intracellular electric field distribution is non-uniform with highest field intensities at the cleavage plane (black lines in the left panel of Figure 1). These maximum intensities are much higher than the applied field and appear for frequencies in the range 100–500 kHz depending on the stage of cytokinesis, i.e., how far the cell division has already progressed. The corresponding curves are plotted in the right panel of Figure 1, where the highest maximum intracellular field strength of ~22 V/cm is observed for the cell in late cytokinesis. Due to the inhomogeneity in the electric field distribution, significant dielectrophoretic (DEP) forces are expected to develop within the cell and these DEP forces are thought to be important factors in the mechanism of action of TTFields []. This DEP force causes the motion of polarizable particles as a result of the interaction of a non-uniform electric field with their induced dipole moment F=p??E []. The DEP force is proportional to the volume of the particle v, its effective polarizability ? and the square of the gradient of the electric field according to: ?F DEP?=1/2?v?Re[?(E˜??)E˜?] using complex phasor notation [,]. Thus, the magnitude of the DEP force component is proportional to the magnitude of the gradient of the squared electric field, |F|????|E|2?? in (V2/m3). The DEP force component showed well-defined peak frequencies at 500, 200, and 100 kHz, respectively, for the three stages considered, from the earliest to the latest stage []. This coincides with the peaks of the maximum electric field inside the cell, which are presented in the right panel of Figure 1. Apart form testing different field intesities, the computational study tested another aspect of TTFields. Namely, it has been shown that the optimal frequency for the inhibitory effect of TTFields is inversely related to cell size [,] and that cell volume is increased in almost all cell lines treated with TTFields []. The simulation results predicted that the above-mentioned peak frequencies decrease and converge as a function of an increasing cell radius. The corresponding maximum values of the DEP force component also decrease with an increasing cell size with equal decay rates for all cytokinesis stages []. In summary, these results obtained by computational modeling confirmed several predicted outcomes of the application of TTFields to biological cells. During metaphase a uniform non-zero Ei is induced. Depending on cell properties, the frequency window of the predicted transition range might be shifted. During cytokinesis, a non-uniform Eiis induced with a substantially increased strength at the cleavage furrow. Frequency-, cell size-, and field-intensity dependences were confirmed. Experimental validation of the predicted induced field strength values would be of great interest. Electric field strengths have typically only been able to be measured inside membranes with voltage dye and patch-clamp techniques. A promising technique by Tyner et al. [] reported the generation of a nanovoltmeter that can report local electric fields in the cell and its use would be ideal to calibrate the strength and local distribution of electric fields in the presence of externally applied AC electric fields. ## 3. Subcellular Electrical Conduction and Electrostatics ### 3.1. Protein Conduction Biological polymers are made up of various proteins, such as actin and tubulin, or nucleic acids as is the case of DNA and RNA. These structures have uncompensated electrical charges when immersed in water but ionic solutions such as the cytoplasm provide a bath of counter-ions that at least partially neutralize the net electric charge. This, however, results in dipolar and higher-moment electric field distributions complicating the situation greatly. Biological water is also believed to create structures with ordered dipole moments and complex dynamics at multiple scales [], which adds to the complexity of subcellular electric field effects. Additionally, free ions endow the cell with conducting properties along well-defined polymeric pathways as well as in a diffusive way. Membranes support strong electric fields (on the order of 105 V/cm), which, due to counter-ion attraction to charged surfaces in solution, result in Debye screening. This causes an exponential decay of these electric fields on a nm scale [] but not their complete disappearance when measured in the cell interior (hence a field strength of 105 V/cm decreases to approximately 0.01 V/cm over 100 nm). The idea that proteins in organisms may have semiconducting properties dates back many decades [,] but protein conductivity has been found to be strongly dependent on the hydration state of proteins []. Electrical properties of cells and their components were promoted by Szent-Györgyi [,], but significant experimental challenges of measuring electric fields and currents at a sub-cellular level impeded progress in this field. The development of more precise experimental tools in the area of nanotechnology holds great promise for rapid progress in the near future [,]. Owing to the fact that there have been many previous reviews of electromagnetic effects in biology [,,,], here we mainly focus on the electrical properties of MTs, actin AFs, ion channels, cytoplasmic ions and DNA with special interest into dynamical electrical properties involving AC fields in the range of 100 kHz. A crucial role of water in the transmission of electrical pulses due to the structure imparted by hydrophilic surfaces [] is also worth noting. Charge carriers related to protein semi-conduction have largely been electrons, protons as well as ions surrounding proteins in physiological solution. AFs and MTs have been implicated in facilitating numerous electrical processes involving ionic and electronic conduction [,] and have been theorized to support dipolar and/or ionic kink-like soliton waves traveling at speeds in the 2–100 m/s range [,]. Due to strong coupling between electrical and mechanical degrees of freedom in proteins, mechano-electric vibrations of MTs have been modeled both analytically and computationally [,]. Electric fields generated by MTs have been modeled extensively and reviewed recently [,,], although experimental measurement of these fields remains extremely difficult, especially in a live cellular environment. ### 3.2. Electrostatic Interactions Involving Charges and Dipoles of Tubulin The net charge on a tubulin dimer depends on pH and changes from +5 at pH 4.5 to 0 at pH 5 and drops to ?30 at pH 8 []. However, in the cytoplasm, a vast majority of electrostatic charges are screened over the distances greater than the Debye length (which varies between 0.6 and 1.5 nm depending on the ionic strength). Therefore, calculating the force due to an electric field of a static electric field with a strength of 1 V/cm acting on a 10 µm-long microtubule, we find from F = qE, with q = 10?13 C for unscreened charges, that results in F = 10 pN assuming the field is largely undiminished when penetrating a cell, which is in general a major oversimplification. This latter issue will be addressed at the end of this review. Even if the force is essentially unchanged, the Debye screening of electrostatic charges means that less than 5% of the charge remains exposed to the field resulting in a net force of at most 0.5 pN, most likely insufficient to exert a major influence on the cytoskeleton. If the field oscillates rapidly, the net force would cancel out over the period of these oscillations, i.e., on a time scale of microseconds or less. The next aspect of MT electrostatics is the effect on the dipole moments of tubulin dimers and of entire MTs. The dipole moment of tubulin (excluding the very flexible and dynamic C-termini which we discuss separately below) has been estimated to be between 566 debye for the ?-monomer and 1714 for the ?-monomer []. However, this is also strongly tubulin-isotype dependent, so these numbers vary a lot between various tubulin isotypes from 500 to 4000 debye []. Note that 1 debye is a unit of electric polarization and is equal to 3.33 × 10?30 Cm. Therefore, taking the dipole moment of a free tubulin dimer as p = 3000 debye as a representative number, we find the interaction energy U with an electric field of E = 1 V/cm, and obtain U = ?pE, and hence U = 10?24 J. This is clearly too small (4000 times smaller than thermal energy kT) to affect the dynamics of an individual tubulin dimer. However, a single MT contains 1625 dimers per 1 µm of its length, so it could eventually accumulate enough net dipole strength to be significantly affected by the field. Unfortunately, this is very unlikely because of the almost perfect radial symmetry of tubulin dipole arrangements in an MT, which has been predicted by a computer simulation []. The individual dipole moments of constituent dimers will almost perfectly cancel out in the radial arrangement of an MT cylinder. There is a small non-cancellation effect along the MT axis but this amounts to less than 10% of the next dipole moment, hence it is doubtful that an entire MT can be aligned in electric fields with intensities lower than 10 V/cm. Unless one uses time-dependent fields (e.g., those used in Reference []), much stronger fields are needed for static effects. To put it another way, the torque ? between a dipole moment of an MT, p, and an external electric field, E, is proportional to their vector product: ? = pxE. For the force to have a meaningful effect on a microtubule, it should exceed 1 pN for lever arm on the order of 1 µm giving a torque of 10?18 Nm. With fields on the order of 1 V/cm, and a dipole moment of 3000 debye per tubulin dimer, even if these dipoles were perfectly aligned, it would result in a 1 µm MT only experiencing a torque of 10?21 Nm, which is approximately 1000 times too low to be of relevance. Various special situations involving electrostatic effects on MTs were calculated earlier []. Note that a force between a charge and an electric field is given by F = qE(x) where E(x) is screened exponentially over the Debye length, which is approximately 1 nm. Hence, a test charge of +5e a distance of 5 nm from the MT surface for a 10-µm MT, experiences a force of 12 pN in water and 1 pN in ionic solution. A tubulin dimer with a dipole of 3000 debye in the vicinity of a microtubule experiences an electrostatic energy of 3 meV. MT-MT interactions due to their net charges with Debye screening accounted for lead to a net force of 9 pN when separated by 40 nm resulting in net repulsion between them. However, at longer distances attractive forces prevail and the corresponding dipole-dipole attraction at 90 nm is only 0.08 pN. The authors of the references [,,] estimated the maximum electrostatic force in the mitotic plate, which was given as F = 6n2 pN per MT where n is the number of elementary charges on each protofilament. Since F is estimated to be 1–74 pN for a typical MT, the estimate is 0.4–3.5 uncompensated elementary charges per protofilament. The range of values of the forces involved is certainly within the realm of possible force requirements for chromosome segregation (about 700 pN per chromosome). ### 3.3. MT Conductivity The building block of an MT is a tubulin dimer, containing approximately 900 amino acid residues with a combined mass of 110 kDa (1 Da is the atomic unit of mass, 1 Da = 1.7 × 10?27 kg). Each tubulin dimer in an MT has a length of 8 nm, along the MT cylinder axis, a width of about 6.5 nm and the radial dimension of 4.6 nm. The inner core of the cylinder, known as the lumen, is approximately 15 nm in diameter. MTs have been predicted to exhibit intrinsic electronic conductivity as well as ionic conductivity along their length []. MTs have a highly electro-negatively charged outer surface as well as C-terminal tails (TTs), resulting in a cloud of counter-ions surrounding them. Experiment and theory demonstrate that ionic waves are amplified along MTs [,]. Since MTs form a cylinder with a hollow inner volume (lumen), MTs have also been theorized to have special conducting properties involving the lumen [] but there has been no direct experimental determination of the electric properties of the MT lumen. Many diverse experiments were performed to date in order to measure the various conductivities of MTs, with a range of results largely dependent on the experimental method, and this has been reviewed elsewhere []. Interestingly, Sahu et al. [,] measured conductivity along the periphery of MTs, where the DC intrinsic conductivities of MTs, from a 200 nm gap, were found to be approximately 10?1 to 102 S/m. Unexpectedly, MTs at certain specific AC frequencies (in several frequency ranges) were found to be approximately 1000 times more conductive, exhibiting astonishing values for the MT conductivities in the range of 103to 105 S/m [,]. Some resonance peaks for solubilized tubulin dimers were reported as: 37, 46, 91, 137, 176, 281, and 430 MHz; 9, 19, 78, 160, and 224 GHz; and 28, 88, 127, and 340 THz. However, for MTs, the corresponding resonance peaks were given as: 120, 240, and 320 kHz; 12, 20, 22, 30, 101, 113, 185, and 204 MHz; and 3, 7, 13, and 18 GHz. Therefore, for MTs there is some overlap with the 100 kHz range indicating a possible independent confirmation of the sensitivity of MT AC conductivity to this electric field frequency range. These authors showed experimental evidence that the high conductivity of the MT at specific AC frequencies only occurred when the water channel inside the lumen of the MT remained intact []. Electro-orientation experiments involving MTs have shown an increased ionic conductivity (0.150 S/m) compared to the buffer solution free of tubulin by as much as 15-fold []. MTs exposed to low frequency AC fields (f < 10 kHz) exhibit a flow motion due to ionic convection. However, for frequencies above 10 kHz this convection effect is absent. Electric fields with intensities above 500 V/cm and frequencies in the range of 10 kHz–5 MHz, are able to orient MTs in solution. As a point of interest, this frequency range overlaps with the range used by Kirson et al. []. However, the intensities of the electric fields used are substantially higher. For instance, a 900 V/cm field with f = 1 MHz was able to align MTs within several seconds []. Impedance spectroscopy enabled the measurements of the dielectric constant of tubulin as ? = 8.41 []. Uppalapati et al. [] exposed taxol-stabilized MT’s in solution to an AC field, which exhibited electro-osmotic and electro-thermal flow, in addition to MT dielectrophoresis effects. Interestingly above f = 5 MHz, electro-hydrodynamic flows were virtually eliminated, and the conductivity of MTs was estimated at 0.25 S/m. Priel et al. demonstrated MTs’ ability to amplify ionic charge conductivity, with current transmission increasing by 69% along MTs [], which was explained by the highly negative surface charge density of MTs that creates a counter-ionic cloud subjected to amplification along the MT axis []. From Priel et al.’s conductance data, the approximate ionic conductivity of MTs is found to be an astonishing 367 S/m []. Below, in the second part of this review, we quantitatively assess AC electric fields on these ionic conductivity experiments, which are expected to be sensitive to the electric field frequencies in the 100 kHz to 1 MHz range. The multiple mechanisms of MT conductance provide ample possibility to explain the varied reports on MT conductivity in the literature. Ionic conductivity along the outer edge of the MT, intrinsic conductivity through the MT itself, and possible proton jump conduction and conductivity through the inner MT lumen have all been suggested. It is conceivable that TTFields may affect ionic conductivities along MTs as is argued below. ## 4. Collective Effects in the Membrane and Cytoplasm ### 4.1. Membrane Depolymerization Effects The electric field across the membrane is on the order of 105 V/cm (0.1 V over 8–10 nm), which is 4–5 orders of magnitude greater than TTFields’ amplitude. Therefore, a direct effect of TTFields on cancer cells’ membrane potential is expected to be very minor. ### 4.2. Ion Channel Conduction Effects Liu et al. [] reported activation of a Na+ pumping mode with an oscillating electric field with a strength of 20 V/cm, which is comparable to the fields of interest in this review, but at a much higher frequency (1.0 MHz) than those of interest. Moreover, neither K+ efflux nor Na+ influx was stimulated by the applied field in the frequency range from 1 Hz to 10 MHz. These results indicate that only those transport modes that require ATP splitting under the physiological condition were affected by the applied electric fields, although the field-stimulated K+ influx and Na+ efflux did not depend on the cellular ATP concentration in the range 5 to 800 µM. Computer simulation of a four-state enzyme electro-conformationally coupled to an alternating electric field [,] reproduced the main features of the above results. Channel densities strongly vary among different neuronal phenotypes reflecting different stabilities of resting potentials and signal reliabilities. In model cell types such as in mammalian medial enthorinal cortex cells, modeled and experimental results match best for an average of 5 × 105 fast conductance Na+ and delayed rectifier K+ channels per neuron []. In unmyelinated squid axons counts can reach up to 108 channels per cell. In model channels such as the bacterial KcsA channel one K+ ion crosses the channel per 10–20 ns under physiological conductances of roughly 80–100 pS [], which is consistent with the frequencies of external electric stimulation mentioned above. This allows for a maximum conduction rate of about 108 ions/s. Estimating the distances between the center of the channel pore and the membrane surface to scale along 5 nm and assuming the simplest watery-hole and continuum electro-diffusion model of channels, this would provide an average speed of 0.5 m/s per ion. Ion transition occurs through a sequence of stable multi-ion configurations through the filter region of the channels, which allows rapid and ion-selective conduction []. The motion of ions within the filter was intensively studied applying classical molecular dynamics (MD) methods (for a summary see Reference []) and density functional studies (e.g., []). MD methods used in these simulations solve Newton’s equations of motion for the trajectory of ions. Time scales for the processes in ion channels can be estimated by the time for translocations (ttr) between two filter sites separated by ~0.3 nm, i.e., 5 × 10?10 s [] and 5 × 10?11 s []. Transition rates (from potential mean force maps and the Kramer transition rate model [] are consistent with these numbers. Changes between a non-conductive and a conductive state in the KcsA occur at a rate of 7.1 × 103 s?1, giving a life-time of the non-conducting state of 0.14 ms (~10?4 s) []. As the duration of the rather (stable) non-conducting state scales in the range 10?3–10?4 s and the within filter translocation time is on the order of 10?11 s, we can expect about 107 filter state changes during a non-conducting state and about 1010 switches per second (10 GHz). Consequently, these time scales are incompatible with those resulting from the effects of 100 kHz electric fields (10 ?s). ### 4.3. Electric Field Effects on Cytoplasmic Ions The cytoplasm provides a medium in which fundamental biophysical processes, e.g., cellular respiration, take place. Most biological cells maintain a neutral pH (7.25–7.35) and their dry matter is composed of at least 50% of protein). The remaining dry material is composed of nucleic acids, trace ions, lipids, and carbohydrates. Most of the trace ions are positively charged. A few metallic ions are found which are required for incorporation into metallo-proteins, e.g., Fe2+, typically at nanomolar concentrations. In Table 1, we summarize the composition of the cytoplasm regarding the most abundant and important components. Composition of the cytoplasm. Based on the above, we can estimate the net force on the total charge in the cytoplasm as F = qE, q = 4 × 1011 e and E = 1 V/cm, so the total force is approximately 6 µN, which is sufficient to cause major perturbations in the cell interior. As discussed above, this is strongly depended on the ability of the electric field to penetrate into the cell’s interior, which is easier in the case of non-spherical cells. The net outcome of these ionic oscillations away and towards attractively interacting protein surfaces inside the cytoplasm can be a concomitant series of oscillations of the structures affected by the ionic clouds as schematically shown below. The viscosity of cytoplasm is approximately ? = 0.002 Pa·s [], hence we can estimate the friction coefficient for an ion in solution as ? = 6??r where r is the ionic radius (hydration shell radius) and find ? = 2 × 10?12 Pa·s·m. In an oscillating electric field of amplitude 1 V/cm and a frequency f = 200 kHz, an ion’s position will follow periodic motion given by: x(t) = 0.1·A·sin(2?ft), i.e., will execute harmonic motion out of phase with the field, with the same frequency and an amplitude A approximately 10% of the radius. However, these ions are simultaneously subjected to the Brownian motion due to their collisions with the molecules of the solvent. To estimate the effect of an oscillating external electric field on the diffusion of a single biomolecular particle (protein, DNA, simple ion, etc.), the Langevin equation can be written down and solved. In the Ito interpretation [], the position Xt of such a particle is given as a function of time by []: dXt= F(Xt)?dt+2kBT???????dWt (1) where ? is the friction coefficient of the particle, T=310 ? is the temperature and kB is the Boltzmann constant. The first term on the RHS of Equation (1) accounts for the influence of deterministic forces F(Xt). Assuming there is no interaction other than the coupling with an external electric field E(Xt), we can write F(Xt)=qE(Xt) where q is the net charge of the particle. At intermediate frequencies, i.e., around 100–200 kHz, the wavelength is around 1000 m, which is obviously much larger than the size of a typical cell. Thus, assuming no important changes due to the dissipation of the field, E can be considered almost constant in a cellular environment: F(Xt)=qE(t). The second term on the RHS represents the random motion, which is due to the many kicks with the surrounding water molecules. Hence, dWt is usually given by []: dWt~dt1/2 ?(t) (2) where ?(t) is a random number, which follows a normal distribution with a mean equal to 0 and a variance equal to 1. Since the Brownian motion is proportional to dt1/2, an estimate of dt is needed to evaluate the influence of the external electric field over the thermal noise. The time step dt can be estimated by the time interval between two series of collisions with water molecules, each series being the sum of enough collisions so that the outcome is approximately Gaussian. In other words, one can assume dt=dx/vH2O, where dx is the typical separation between two water molecules, i.e., dx=mH2O/?H2O??????????3 where mH2O is the mass of one water molecule and ?H2O is the mass density of water. Here, vH2O is the velocity of water molecules given by vH2O=3kBT/mH2O???????????. The use of the above parameters leads to a typical time step of dt~5.0×10?13 s. The two terms in the RHS of Equation (1) above can be compared to estimate the effect of an electric field over the thermal noise. In the case of a spherical particle, we can assume ?=6??r, where the hydrodynamic radius is r=1.8 ? and the viscosity of the cytoplasm is ?=0.002 Pa·s []. By taking q=1 e (a single ion) and E=E0cos2?ft with E0=1 V/cm, it turns out that the amplitude of the coupling term associated with the electric field is qE0/?=2.36 × 10?6 m/s. On the other hand, the noise coefficient is 2kBT/???????? (dt)?1/2=50.2 m/s when the estimate obtained above is used: dt~5.0×10?13 s, which is much larger than the deterministic term. Even in the case of less frequent Brownian collisions, e.g., dt~10?6 s, the noise coefficient is 0.035 m/s which is still much larger than the coupling with the electric field, meaning that an electric field of amplitude 1 V/cmhas an exceedingly small probability to influence the diffusion of a single Brownian particle even if the net charge q is 100–1000 times larger as in the case of a protein. Alternatively, it can be shown that an oscillating electric field at intermediate frequencies with an amplitude of 1 V/cm has no direct sizable effect on the diffusion of biomolecules by considering an ensemble of molecules instead of a single Brownian particle. Assuming a constant electric field E, the distribution of particles as a function of time is given by []: P(x,t)= 12Dt????exp?????(x x0?qEt?)22Dt???? (3) Here, D=kBT/? is the diffusion coefficient for one particle. From the above equation, a typical time when the particles start to be drifted away because of the electric field is t=2(kBT)?/(qE)2. For a single ion (q=1 e, r=1.8 ?), t=226.1 s, whereas for a typical globular protein (q~100 e, r~1.0 nm), t=0.13 s, which is much larger than the period of an electric field oscillating at hundreds of kHz. For the sake of simplicity, we have not discussed here how an electric field could induce conformational changes in biomolecular structures, which would affect their charge distributions and dipolar spectra, which, in turn, could modify their diffusion by inducing new interactions with the surrounding molecules. An estimate of such indirect effects would require careful investigations of the studied system based on realistic MD simulations. In this case, the external electric field can be either computationally modeled by initializing the system with added kinetic energy in the directions of the normal modes or by adding an extra coupling term to the force field []. ## 5. AC Electric Field Effects on Subcellular Structures ### 5.1. Electric Field Effects on MTs Several experimental efforts were made aimed at measuring the electric field around MTs. Vassilev et al. [] observed alignment of MTs in parallel arrays due to the application of electric fields with intensities of 0.025 V/cm and of pulsed shape. In cell division, coherent polarization waves have been implicated as playing the key role in chromosome alignment and their subsequent separation [,]. Electric fields in the range of 3 V/cm were applied by Stracke et al. [] to suspended MTs, which moved at pH 6.8 from the negative electrode to the positive one indicating a negative net charge, and an electrophoretic mobility of about 2.6 × 10?4 cm2·V?1·s?1. The work of Uppalapati et al. [] covers the range of frequencies overlapping with TTFields, although the amplitudes are much larger due to the voltage bias of 40 V across a 20-µm gap giving an electric field of 2 × 104 V/cm as opposed to 1 V/cm). Below 500 kHz, MTs flow toward the centerline of electrodes. The electro-osmotic force causes the movement of the fluid in a vortex-like manner. This represents the Coulomb force experienced by the ionic fluid due to the applied voltage. The fluid flow velocity ? is proportional to the tangential component of the electric field Et, surface charge density ?, the solution’s viscosity ? and the inverse Debye length ? such that: ? = Et ?/??. At lower frequencies, flow velocity is larger. On the other hand, due to strong heating effects of the AC field, the electro-thermal force causes motion of MTs along the length of the electrodes. Above 500 kHz MTs flow toward the gap between the electrodes due to dielectrophoresis. The DEP force experienced by MTs in a non-uniform electric field is given by: ?FDEP?=14??m[?2?m(?p??m)+?m(?p??m)?2?2m+?2m]?|E|2 (4) where the symbols with subscript “m” refer to the medium and “p” to the particle. Hence, this process is largely driven by the difference between the conductivities and permittivities of the MTs and the medium, (?p ? ?m) and (?p ? ?m), respectively. We predict that lowering the pH of the solution to the isoelectric point of MTs around pH 5 should substantially reduce this effect and additionally lowering the frequency will reduce it further due to the dependence of the first term on the square of the frequency. At ~5 MHz, the electro-osmotic and electro-thermal flow balance each other out with the flow of MTs being solely due to dielectrophoresis. It is important to compare the dielectrophoretic force to Brownian motion in order to determine whether or not electric fields are sufficiently strong to overcome random motion, i.e., to find out if the dielectric potential exceeds the thermal energy, i.e., ?r3?m[(?p??m)(?p+2?m)]E2>kT (5) where ?m is the dielectric constant of the medium and ?p is the dielectric constant of the particle. E is the electric field strength and r the radius of the particle. Taking as an example a tubulin dimer in solution and the corresponding values of the dielectric constants, one finds that E must exceed 0.25 V/cm for the field to be effective in orienting polarizable tubulin dimers. Similarly, for a 10-µm long MT we replace the factor ?r3 with ?r2 L, where r is the radius of a MT (12.5 nm) and L its length, to obtain a condition that E > 0.01 V/cm. Clearly, the electric field values of 1 V/cm (even if they are screened by a large factor inside the cell) are sufficient to exert electrophoretic effects on tubulin and MTs. The longer the MT, the more pronounced the dielectrophoretic effect is predicted to occur. Recently, Isozaki et al. [] used MTs labeled with dsDNA to manipulate the amount of net charge and observe the mobility of these hybrid structures compared to control where MTs where only labeled fluorescently with two different tags. It was found for control MTs that the electrophoretic mobility is approximately: 2 × 10?8 m2·V?1·s?1which is consistent with Stracke et al. []. For field strengths of approximately 1 V/cm, one can estimate the average velocity of MT translocations as 2 µm/s. They also stated ?D = 0.74 nm as the Debye length, ? = 8.90 × 10?4 kg·m?1·s?1 and ? = 6.93 × 10?10C·V?1·m?1 as the viscosity and dielectric constant of the buffer, respectively. Importantly, they estimated the effective charges of the TAMRA- and AlexaFluor 488-tagged tubulin dimer as 10 e? and 9.7 e?, which obviously is only a fraction (approximately 20%–30%) of the vacuum values but much larger than earlier experimental estimates. Electrophoresis experiments were also performed by van den Heuvel et al. [], with electric field strengths of 40 V/cm, yielding MT electrophoretic mobility in the range of 2.6 × 10?8 m2·V?1·s?1, in line with previous reports. They found the effective charge of a tubulin dimer to be approximately 23 e?. ### 5.2. Tubulin’s C-Termini Dynamics and AC Electric Fields Computer simulations demonstrate that ionic waves can trigger C-termini to change from upright to downward conformations initiating propagation of a travelling wave []. This wave is predicted to travel as a “kink” solitary wave with a phase velocity of vph = 2 nm/ps []. A typical time scale for C-termini motion is 100 ps, which is too fast for the 100 kHz frequency range of TTFields. However, C-termini being very flexible and highly charged (with approximately 40% of the tubulin’s charge located there) are likely to dynamically respond to electric fields as local changes of pH are correlated with positive and negative electric field’s polarities, respectively. This effect can cause MT instability as well as interference with motor protein transport as discussed below. A stable dimer conformation is predicted to have C-termini cross-linked between the monomers as shown in Figure 2. A cross-linked conformation of C-termini stabilizes a straight orientation of a tubulin dimer. A disruption of this conformation can cause MT instability. ### 5.3. Ionic Waves along MTs and AC Electric Fields Manning [] postulated that polyelectrolytes may have condensed ions in their surroundings if a sufficiently high linear charge density is present on the polymer’s surface []. The Bjerrum length, ?B, is defined as the distance at which thermal fluctuations are equally strong as the electrostatic interactions between charges in solution whose dielectric constant is ? at a given temperature T in Kelvin. Here, ?0denotes the permittivity of the vacuum and kB is the Boltzmann constant. Counter-ion condensation occurs when the average distance between charges, b, is such that ?B/b = S> 1. In this case, the cylindrical volume of space depleted of ions outside the counter-ion cloud surrounding the polymer functions as an electrical shield. The “cable-like” electro-conducting behavior of such a structure is supported by the polymer itself and the “adsorbed” counter-ions, which are “bound” to the polymer in the form of an ionic cloud (IC). Tuszynski et al. [] calculated an electrostatic potential around tubulin and extended this to an MT, which demonstrated non-uniformity of the potential along the MT radius with periodically repeating peaks and troughs along the MT axis. Consequently, MTs have been viewed as “conducting cables” composed of 13 parallel currents of ionic flux (corresponding to 13 protofilaments of MTs) and attracting an IC of positive counter-ions close to its surface and along tubulin C-terminal tails (TT), while negative ions of the cytosol are repelled away from the MT surface. The thickness of the negative ion depleted area corresponds to the Bjerrum length. An estimate of the respective condensate thickness ? of the counter-ion sheath for the tubulin dimer (?TD) and C-termini (?TT) is ?TD = 2.5 nm and ?TT = 1.1 nm, as analyzed in []. Using a Poisson–Boltzmann approach, the capacitance of an elementary ring of an MT consisting of 13 dimers is found as []: C0=2??0?lln(1+lBRIC) (6) where l stands for the length of a polymer unit and RIC = ?TD + ?TT for the outer radius of an IC. For a tubulin dimer: CTD = 1.4 × 10?16 F and for an extended TT: CTT = 0.26 × 10?16 F. Hence: C0=C0+2×C0=1.92×10?16 F (7) Estimating the electrical resistance for a complete tubulin ring gives R0 = 6.2 × 107 ? [,]. Including the conductance of both nanopores through an MT surface accounts for the leakage of IC cations into the lumen area and gives a conductance G0, of a ring as G0 = ?1 + ?2 = (2.93 + 7.8) nS = 10.7 nS and the corresponding resistivity as R = 1/G0 = 93 M?. A simple equivalent periodic electric circuit simulating one protofilament of an MT consists of a long ladder network composed of elementary circuit units as shown in Figure 3 []. An effective circuit diagram for the n-th unit with characteristic elements for Kirchhoff’s laws applied to a microtubule as an ionic cable []. The longitudinal ionic current encounters a series of Ohmic resistors R0 for each ionic conduction unit (an MT ring). The nonlinear capacity with the charge Qn for the n-th site of the ladder is in parallel with the total conductance G0 of the two TTs of a dimer. Then using Kirchhoff’s law: in?in+1=?Qn?t+G0?n, (8) ?n?1??n=R0in, (9) we find the equations for the voltage propagation: ?Qn?t=C0??n?t?C0?0??n?C0?0?(t?t0)??n?t?2b0C0?n??n?t (10) Introducing an auxiliary function u(xt) unifying the voltage and its accompanying IC current as: un=Z1/2in=Z?1/2?n (11) with the characteristic impedance defined as: Z=1?C0, (12) leads in the continuum limit to the electric signal propagation equation: ?2?u?x?l23?3u?x3?ZC0l?u?t+ZC0?0?l(t?t0)?u?t+2Z3/2b0C0lu?u?t?1l(ZG0+Z?1R0?ZC0?0?)u=0 (13) The characteristic charging (discharging) time of an elementary unit capacitor C0through the resistance R0 is given by T0 = R0C0 with an estimate for T0 = 1.2 × 10?8 s and the characteristic propagation velocity of the ionic wave: v=l/T0 as v0 = 0.67 m/s. A standard travelling-wave with speed v, for the normalized function u(xt), can be used as a solution of the propagation equation, which is a soliton that preserves its width but its amplitude decays over the length of about 400 units corresponding to 3.2 µm, which is of the order of the MT length. Interestingly, a characteristic time for this excitation can readily be estimated as 1.2 × 10?5 s whose inverse, the frequency, f, is very close to the TTField value, i.e., 90 kHz. The maximum frequency allowed in this model is 68 MHz. To summarize, ionic conduction along and away from charged protein filaments such as MTs involves cable equations resulting from equivalent RLC circuits surrounding each protein unit in the network. Conduction along the filaments experiences resistance due to viscosity in the ionic fluid. Capacitance is caused by charge separation forming a double layer between the MT surface and ions with a distance separating them comparable to the Bjerrum length. Inductance is caused by helical nature of the MT surface and consequently, solenoidal flows of the ionic fluid along and around the MT. The key numerical estimates of the RLC circuit components are as follows []. For a single dimer: C = 6.6 × 10?16 F, R1 = 6 × 106 ? (along the MT), R2 = 1.2 × 106 ? (perpendicular to the MT) and L = 2 × 10?12 H. These numbers can be used to estimate characteristic time scales for the oscillations (LC) and exponential decay (RC) taking place in this equivalent circuit. We obtain for decay times (? = RC) the following values: (a) ?1 = 10?8 s along the MT length and (b) ?2 = 10?9 s away from the MT surface. However, due a low value of inductance L, the corresponding time for electromagnetic oscillations is found using ?0 = (LC)1/2 as ?0 = 0.2 × 10?12 s = 0.2 ps. Clearly, the oscillation times are too short for potential effects with 100 kHz-range fields (the time of TTFields oscillations is on the order of 5–10 µs). The decay times are much closer so we will focus on these parameters. Repeating these calculations for a microtubule of length l, we note that R1 scales with length of a microtubule, while R2 is length independent. The corresponding capacitance in both cases scales with length, therefore ?1 scales with length squared (l2) while ?2 scales with length. To obtain actual values, we need to multiply the values for a single ring by the number of rings in an MT. We use the values found for a single ring, i.e., ?1 = 10?8 s and ?2 = 2 × 10?9 s and scale them accordingly to estimate the length of MTs that could experience resonant effects in terms of ionic currents along and away from their surface. This way we find the scaling factor that leads to the characteristic times on the order of 10 µs. Therefore, for longitudinal effects, on the order of 50 rings, MTs only 400 nm long would respond to 100 kHz stimulation. On the other hand, for ionic flows pulsating radially around an MT, a 20-µm long MT would be required. These results are very sensitive regarding the choice of parameter values, especially the resistivity where diverse estimates can be found in the literature. In general, there is strong overlap between the time scales of ionic wave propagation and electric field stimulation. It is conceivable that both effects play a role depending on the orientation of the field vis a vis the geometry of mitotic spindles and the MTs forming them. It appears that short MTs would be more sensitive to the longitudinal wave generation by TTFields while long MTs should lead to perpendicular wave generation. Current densities should also be briefly discussed in relation to previously reported endogenous current densities, j, in cells, which range from 0.2 to 60 µA/ cm2 []. This translates into 0.002 < j < 0.6 A/m2. Since j = ?E where E = 1 V/cm and ? of the cytoplasm has a large range of values reported between 0.1 and 100, we see that even taking the lower limit of 0.1 would result in ionic currents along MTs that would overwhelm the intrinsic ion flows in a dividing cell. It is possible that these externally stimulated currents cause a major disruption of the process of mitosis and associated intra-cellular effects. It is also worth mentioning that recently metabolic oscillations in cells with a period of approximately 10 to 12 s, were measured in vivo [] which is many orders of magnitude slower than any AC electric field effects discussed here. Hence, it is safe to assume that there is a very unlikely possibility of electric field effects in the 100 kHz range to interfere with cellular metabolism. Finally, it is interesting to address the issue of the power dissipated due to a current flowing along an MT. Again, we take as an example a 10 µm-long MT, and we estimate the average power drain as: ?P?=(1/2)V20[R/(R2+X2c)], (14) where Xc= 1/?C is the capacitive resistance. Substituting the relevant numbers we obtain the power dissipated to be in the 10?11 W range which is comparable to the power generated by the cell in metabolic processes (100 W of power generation in the body/3 × 1013 cells in the body). Consequently, additional heat generated by these processes may be disruptive to living cells although there is no experimentally detected thermal effect of TTFields. ### 5.4. Resonance Effects on MTs Cosic et al. [,] reported EM resonances in biological molecules (proteins, DNA and RNA) in THz, GHz, MHz and kHz ranges. They proposed the so-called resonant recognition model (RRM) based on the distribution of energy of delocalized proteins in a biological system and charge transfer under resonance with a velocity of 7.87 × 105m/s and covering distances of 3.8 Å between amino acids, giving a characteristic frequency between 1013 and 1015 Hz. Then they state a variety of charge transfer velocities yielding different resonant frequencies. Of particular interest to this review is the velocity v = 0.0005 m/s which produces EMF in the range of 108–325 kHz for TERT, TERT mRNA and Telomere. This velocity corresponds the propagation of solitons on ?-helices. For tubulin and MTs, three specific ranges of resonant frequencies have been predicted by the RRM approach: 97–101 THz, 340–350 THz and 445–470 THz, none of which overlaps with TTField frequencies. H-bond strength in MTs has been recently computationally estimated [] as ranging from 11.9 k/mol for the weakest bond to 42.2 kJ/mol for the strongest one and a total of 462 kJ/mol for the ?-tubulin/?-tubulin interactions and 472 kJ/mol for the ?-tubulin/?-tubulin interactions, which based on the Planck relationship between frequency and energy translates into a range of frequency values between 0.3 × 1014 Hz and 1.3 × 1015Hz. Again, these frequencies are much too high to be affected by TTFields. Therefore, we do not expect TTFields to be capable of disrupting the MT structure. Furthermore, Pizzi et al. [] measured microwave resonance effects in MTs and found a resonant frequency at 1.510 GHz. This may not correspond to bond-breaking between tubulin dimers but simply to some specific electro-mechanical oscillations. Finally, Preto et al. [] re-evaluated the Froehlich mechanism for long-range interactions and concluded that classical electromagnetic dipole-dipole interactions at high enough frequencies can lead to attraction between oscillating dipoles over distances comparable to the size of the cell. However, even including a coherently coupled layer of water molecules around a protein, this would require frequencies in the THz range or higher. Consequently, almost all of the resonant frequencies listed above fall well outside the range of potential overlap with the 100 kHz frequencies of TTFields. ### 5.5. Ionic Wave Conductivity along Actin Filaments and AC Fields AFs are approximately 7 nm in diameter, with a periodic helical structure repeating every 37 nm. Actin filaments are arranged from actin monomers resulting in an alternating distribution of electric dipole moments along the length of each filament []. They are characterized by a high electrostatic charge density [,] resulting in ionic current conductivity involving the counter-ions surrounding them [], which is very similar to the effects observed for MTs []. The observed wave patterns in electrically-stimulated AFs [] were very similar to the solitary waveforms recorded for electrically-stimulated non-linear transmission lines []. In these experiments [,], an input voltage pulse was applied with an amplitude of 200 mV for a duration of 800 ms. Electrical signals were measured at the opposite end of the AF demonstrating that AFs support axial non-linear ionic currents. Since AFs produce a spatially-dependent electric field arranged in peaks and troughs [] with an average pitch ~35–40 nm, they can be modeled as an electrical circuit with the following non-linear components: (a) a non-linear capacitor associated with the spatial charge distribution between the ions located in the outer and inner areas of the polymer; (b) an inductor; and (c) a resistor, similar to the model described above developed for MTs. A helical distribution of ions winding around the filament at an approximate distance of one Bjerrum length to the filament corresponds to a solenoid in which an ionic current flows due to the voltage gradient between the two ends. For an AF with n monomers, its effective resistance, inductance, and capacitance are, respectively: Reff=(?ni=11R2,1)?1+?ni=1R1,i, (15) Leff=?ni=1Li, (16) Ceff=?ni=1C0,i, (17) where R1,i = 6.11 × 106 ?, and R2,i = 0.9 × 106 ?, such that R1,i = 7R2,i []. Hence, for a 1-µm length of an AF we find that Reff = 1.2 × 109 ?, Leff = 340 × 10?12 H and Ceff = 0.02 × 10?12 F. The electrical model of an AF is an application of Kirchhoff’s laws to one section of the effective electrical circuit that is coupled to neighboring monomers. In the continuum limit [] the following equation describes the spatio-temporal behavior of the electric potential propagating along the actin filament: LC0?2V?t2=a2(?xxV)+ R2C0??t(a2(?xxV))? R1C0?V?t+R1C02bV?V?t. (18) Solitary ionic waves have been described as the solutions of the above nonlinear partial differential equation [] with an estimated velocity of propagation between 1 and 100 m/s []. This model has been recently updated with a more plausible estimation of model parameters []. Like MTs [], AFs can be manipulated by external electric fields []. In a similar manner to our analysis of the time scales for MTs as ionic conduction cables with RLC components, we estimate similar time scales for actin and AFs. We readily find for a single actin monomer, that the time scale for LC oscillations is very fast, namely ?0 = (LC)1/2 and ?0 = 6 × 10?14 s. Secondly, the decay time for longitudinal ionic waves is ?1 = R1C = 6 × 10?10 s while the corresponding time for radial waves is ?2 = R2C = 0.9 × 10?10 s. All of the above time scales are not compatible with interactions involving electric fields in the 100 kHz range. However, the situation changes drastically for AFs where there is a similar scaling with the length of the filament as described above for MTs. Taking as an example a 1-µm AF, we find ?0 = 10?11 s, which is still too short but ?1 = R1C = 2.4 × 10?5 s which is in the correct range of time for interactions with AC electric fields in the 100 kHz range. It should be noted that AFs have been found sensitive to AC fields under experimental conditions []. ### 5.6. Electric Field Effects on DNA Anderson and Record [] described ionic distribution around DNA in great detail. During interphase, DNA contents present in the nucleus are expected to be protected from external fields due to being enclosed in the nearly spherical nuclear membrane []. In addition to the screening effects of being shielded both by the cell membrane and the nuclear wall, the irregular geometry of the DNA strands and their short persistence length indicate that while highly charged, DNA is unlikely to participate in ionic conduction effects shown either for AFs or MTs, both of which have very large persistence lengths. However, at the beginning of mitosis, the nuclear membrane breaks down, thus potentially not shielding the DNA any longer which would allow for the action of electric fields on chromosomes. ### 5.7. Electric Field Effects on Motor Proteins Kinesin participates in mass transport along MTs and propagates at a maximum speed of 10?6 m/s. This value depends on the concentration of ATP and the ionic concentrations in the medium. In the case of MTs, kinesin transports various crucial cargo and for actin filaments, dynein does the same at similar speeds. Hence each step of a motor protein takes place over the period of a few ms, which is much longer than the period of AC field oscillations. However, kinesin binds to MTs through C-termini, which are very sensitive to electric field fluctuations and hence it is possible that kinesin transport would be very strongly disrupted by these rapid oscillations of C-termini. This aspect merits careful experimental verification. Another potential member of the cytoskeleton that has been found affected by TTFields [] is the protein called septin, which are GTP-binding like tubulin but form oligomeric hetero-complexes including rings and filaments. There is no information at the present time that could shed light on the mechanism of TTField effects with septin-based structures. ## 6. Discussion The cytoskeleton and especially, MTs, may participate in numerous interactions with electromagnetic forces due to the complex charge distribution in and around these protein filaments surrounded by poly-ionic solutions. First of all, there are large net charges on tubulin, which are largely but not completely screened by counter-ions. Secondly, some of the charges are localized on C-termini, which are very flexible leading to oscillating charge configurations. Then, there are ions surrounding the protein that can be partially condensed and susceptible to collective oscillations. Moreover, there are large dipole moments on tubulin and microtubules whose geometric organization importantly affects their response to external fields. Finally, there can be induced dipole moments especially in the presence of electric field gradients. Disentangling the relative importance of the various effects under different conditions is not trivial and requires careful examination. Depending on the orientation of the electric fields with the cell axis and in particular with the MT axis (however, they fan out from centrosomes in mitotic cells, so there will be at different angles to any field), there could in general be three types of ionic waves generated: 1. Longitudinal waves propagating along the MT surface. In this case each protofilament of a microtubule acts like a cable with its inherent resistance r, so the resistance of an entire microtubule would be R = r/13 since all these cables are in parallel to each other. 2. Helical waves propagating around and along each microtubule, there could be three or five such waves propagating simultaneously mimicking the three-start or five-start geometry of a microtubule. The effective resistance of such cables would be the individual resistance divided by the number of cables in parallel. 3. Radial waves propagating perpendicularly to the microtubule surface. If a field is oriented at an angle to the MT axis, it is expected that all these wave types may be generated simultaneously. Once AC fields generate oscillating ionic flows, these can in turn: 1. Interfere with ion flows in the cleavage area of dividing cells. 2. Interfere with motor protein motion and MAP-MT interactions. 3. May to a lesser degree affect ion channel dynamics. 4. May in general affect the net charge of the cytoplasm. Finally, Kirson et al. [] mention intracellular charged and polar entities such as cytoplasmic organelles as being potentially most directly affected by TTFields. This is not specifically addressed in this paper due to size and scope limitations as well as the scarcity of data in this regard. It has been argued [] that inhomogeneity in field intensity may exert a uni-directional electric force on all intracellular charged and polar entities, pulling them toward the furrow (regardless of field polarity). It was determined that cytoplasmic organelles are electrically polarized by the field within dividing cells. As a consequence, the TTField-generated forces acting on these organelles may reach values up to 60 pN resulting in their movement toward the cleavage furrow. These organelles can move at velocities up to 30 ?m/s and, as a result, they could pile up at the cleavage furrow within a few minutes, interfering with cytokinesis, which may lead to cell destruction. This aspect needs detailed experimental investigation. Some measurable heating effects in the cytoplasm might also be expected. These fields are not expected to affect permanent dipoles of proteins such as tubulin and actin. Although TTField effects have not been specifically assessed for AFs, an earlier paper [] investigated exposure of cells to AC electric fields in a low frequency range of 1–120 Hz and found significant induced alterations in the AF structure, which were both frequency- and amplitude dependent. An application of 1–10 Hz AC fields caused AF reorganization from continuous, aligned cable structures to discontinuous globular patches. Cells exposed to 20–120 Hz electric fields were not visibly affected. The extent of AF reorganization increased nonlinearly with the electric field strength. The characteristic time for AF reorganization in cells exposed to a 1 Hz, 20 V/cm electric field was approximately 5 min. Importantly, applied AC electric fields were shown to initiate signal transduction cascades, which in turn cause reorganization of cytoskeletal structures. Therefore, in addition to direct effects of TTFields, there may be indirect, down-stream interactions. ## 7. Conclusions Based on the extensive analysis of the various possible effects AC electric fields can have on living cells, we conclude the following. Electric field gradients, especially in dividing cells, cause substantial DEP forces on tubulin dimers and MTs. The longer the MT, the more pronounced the effect. Additionally, another likely scenario is that ionic current flows along and perpendicular to MT surfaces (as well as actin filaments, but less likely) take place, which can be generated by AC field oscillations in the 100–300 kHz range. The specific frequency selection depends critically on the length of each filament. Identification of the strength, cause, and function of intracellular electric fields has only recently been experimentally accessible, although speculations in this area have existed for over a decade. These insights may also assist in devising and optimizing ways and means of affecting cells, especially cancer cells, by the application of external electric fields. With the advent of nanoprobe technology, which has shown promise in measuring these fields at a subcellular level, it is very timely to explore the various physical properties of the cytoplasmic environment including the cytoskeleton and the ionic contents of the cytoplasm. This research promises to contribute to our understanding of the cytoplasm in live cells and the role of microtubules and mitochondria in creating dynamic and structural order in healthy functioning cells. It will also be of help to identify biophysical differences in cancer cells that lead to increased metastatic behavior. Such an understanding may lead to optimized therapies and the identification of specific targets to halt metastatic transformation, as well as insights into the mechanism of action of current electromagnetic cancer therapies that are FDA approved and are in development. ## Acknowledgments Cornelia Wenger was supported by Novocure. Douglas E. Friesen was supported by Novocure. Douglas E. Friesen also gratefully acknowledges support from Alberta Innovates Health Solutions and the Alberta Cancer Foundation. The funding for J.A.T.’s research comes from the Natural Sciences and Engineering Research Council of Canada. ## Abbreviations The following abbreviations are used in this manuscript:  DC direct current AC alternating current TTFields Tumor Treating Fields GBM glioblastoma multiforme EM electromagnetic MT microtubule DEP dielectrophoretic AF actin filament TT C-terminal tail MAP microtubule associated protein ## Author Contributions Jack A. Tuszynski produced the first draft of the manuscript. Cornelia Wenger performed the computational studies and contributed to editing the paper. Douglas E. Friesen helped conceive the ideas presented in the paper and contributed to editing the paper. Jordane Preto contributed the analysis of ion motion in electric fields. ## Conflicts of Interest Novocure had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. ## References 1. 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Chem. 1990;19:4232–4265. doi: 10.1146/annurev.bb.19.060190.002231.[PubMed] Articles from International Journal of Environmental Research and Public Health are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI) ## Laser and NSAIDs Lasers Med Sci. 2017 Aug 9. doi: 10.1007/s10103-017-2299-2. [Epub ahead of print] # Effects of photobiomodulation therapy and topical non-steroidal anti-inflammatory drug on skeletal muscle injury induced by contusion in rats-part 2: biochemical aspects. Tomazoni SS1, Frigo L2, Dos Reis Ferreira TC3,4, Casalechi HL3, Teixeira S5, de Almeida P6, Muscara MN5, Marcos RL6, Serra AJ6, de Carvalho PTC4,6, Leal-Junior ECP3,4. ### Author information 1 Masters and Doctoral Programs in Physical Therapy, Universidade Cidade de São Paulo (UNICID), Rua Cesário Galeno, 448/475, São Paulo, SP, 05508-900, Brazil. shaiane.tomazoni@gmail.com. 2 Biological Sciences and Health Center, Cruzeiro do Sul University (UNICSUL), São Paulo, SP, Brazil. 3 Laboratory of Phototherapy in Sports and Exercise, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil. 4 Postgraduate Program in Rehabilitation Sciences, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil. 5 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. 6 Postgraduate Program in Biophotonics Applied to Health Sciences, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil. ### Abstract Muscle injuries trigger an inflammatory process, releasing important biochemical markers for tissue regeneration. The use of non-steroidal anti-inflammatory drugs (NSAIDs) is the treatment of choice to promote pain relief due to muscle injury. NSAIDs exhibit several adverse effects and their efficacy is questionable. Photobiomodulation therapy (PBMT) has been demonstrated to effectively modulate inflammation induced from musculoskeletal disorders and may be used as an alternative to NSAIDs. Here, we assessed and compared the effects of different doses of PBMT and topical NSAIDs on biochemical parameters during an acute inflammatory process triggered by a controlled model of contusion-induced musculoskeletal injury in rats. Muscle injury was induced by trauma to the anterior tibial muscle of rats. After 1 h, rats were treated with PBMT (830 nm, continuous mode, 100 mW of power, 35.71 W/cm2; 1, 3, and 9 J; 10, 30, and 90 s) or diclofenac sodium (1 g). Our results demonstrated that PBMT, 1 J (35.7 J/cm2), 3 J (107.1 J/cm2), and 9 J (321.4 J/cm2) reduced the expression of tumor necrosis factor alpha (TNF-?) and cyclooxygenase-2 (COX-2) genes at all assessed times as compared to the injury and diclofenac groups (p < 0.05). The diclofenac group showed reduced levels of COX-2 only in relation to the injury group (p < 0.05). COX-2 protein expression remained unchanged with all therapies except with PBMT at a 3-J dose at 12 h (p < 0.05 compared to the injury group). In addition, PBMT (1, 3, and 9 J) effectively reduced levels of cytokines TNF-?, interleukin (IL)-1?, and IL-6 at all assessed times as compared to the injury and diclofenac groups (p < 0.05). Thus, PBMT at a 3-J dose was more effective than other doses of PBMT and topical NSAIDs in the modulation of the inflammatory process caused by muscle contusion injuries. Med Oral Patol Oral Cir Bucal. 2017 Jul 1;22(4):e467-e472. # Effect of pre-operatory low–levellaser therapy on pain, swelling, and trismus associated with third-molar surgery. Petrini M1, Ferrante M, Trentini P, Perfetti G, Spoto G. ### Author information 1 Department of Medical, Oral and Biotechnological Sciences, University of Chieti – Italy, Via Vestini 31, 66013 Chieti, Italy materialidentari.uda@gmail.com. ### Abstract #### BACKGROUND: The extraction of impacted third molars is commonly associated to pain, edema, trismus, limited jaw opening and movements. The aim of this retrospective study is to verify if pre-surgical lowlevel laser therapy (LLLT) associated with the extraction of impacted lower third molars could add benefits to the postoperative symptoms respect LLLT performed only after surgery. #### MATERIAL AND METHODS: Data from 45 patients subjected to a surgical extraction of lower third molars were pooled and divided into three groups. Patients that received only routine management were inserted in the control group. Group 1, were patients that received LLLT immediately after surgery and at 24 hours. In group 2 were included patients treated with LLLT immediately before the extraction and immediately after the end of the procedure. Data were analyzed using linear regression and descriptive statistics. #### RESULTS: Both laser-treated groups were characterized by minor events of post-surgery complications of pain, edema, trismus. The use of NSAIDs in the first 24 hours was significantly inferior in Group 2. #### CONCLUSIONS: Pre-surgical LLLT treatment seems to increase the analgesic effect of LLLT. However, trismus and edema were reduced in both laser treated groups, independently from the period of irradiation. Lasers Med Sci. 2016 Winter;7(1):45-50. doi: 10.15171/jlms.2016.10. Epub 2016 Jan 7. # LowLevelLaser Therapy Versus Pharmacotherapy in Improving Myofascial Pain Disorder Syndrome. Khalighi HR1, Mortazavi H1, Mojahedi SM2, Azari-Marhabi S1, Moradi Abbasabadi F3. ### Author information 1 Department of Oral and Maxillofacial Medicine, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 Department of Laser, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran,Iran. 3 Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Qom University of Medical Sciences, Qom, Iran. ### Abstract #### INTRODUCTION: Temporomandibular disorders (TMD) lead to masticatory muscle pain, jaw movement disability and limitation in mouth opening. Pain is the chief complaint in 90% of the TMD patients which leads to disability and severe socioeconomic costs. The purpose of this study was to evaluate the therapeutic effects of low level laser therapy (LLLT) compared to pharmacotherapy with NSAIDs (naproxen) in myofascial pain disorder syndrome (MPDS). #### METHODS: In this randomized controlled clinical trial, 40 MPDS patients were divided into two groups. One group received naproxen 500 mg bid for 3 weeks as treatment modality and also had placebo laser sessions. The other group received active laser (diode 810 nm CW) as treatment and placebo drug. Pain intensity was measured by visual analogue scale (VAS) and maximum painless mouth opening was also measured as a functional index every session and at 2 months follow up. Data was collected and analyzed with SPSS software. Independent t test was used to analyze the data. A P < 0.05 was considered significant. #### RESULTS: Low level laser caused significant reduction in pain intensity (P < 0.05) and a significant increase in mouth opening. In naproxen group neither pain intensity nor maximum mouth opening had significant improvement. Pain relief, in subjective VAS was observed in third session in LLLT group, but did not occur in naproxen group. Maximum mouth opening increased significantly in laser group compared to the naproxen group from the eighth session. #### CONCLUSION: Treatment with LLLT caused a significant improvement in mouth opening and pain intensity in patients with MPDS. Similar improvement was not observed in naproxen group. Lasers Med Sci. 2017 Jan;32(1):101-108. doi: 10.1007/s10103-016-2091-8. Epub 2016 Oct 10. # Effects of photobiomodulation therapy, pharmacological therapy, and physical exercise as single and/or combined treatment on the inflammatory response induced by experimental osteoarthritis. Tomazoni SS1, Leal-Junior EC2, Pallotta RC3, Teixeira S3, de Almeida P3, Lopes-Martins RÁ4. ### Author information 1 Laboratory of Pharmacology and Experimental Therapeutics, Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes, 1524, Butantan, São Paulo, SP, 05508-900, Brazil. shaiane.tomazoni@gmail.com. 2 Postgraduate Program in Biophotonics Applied to Health Sciences and Post Graduate Program in Rehabilitation Sciences, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil. 3 Laboratory of Pharmacology and Experimental Therapeutics, Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes, 1524, Butantan, São Paulo, SP, 05508-900, Brazil. 4 Biomedical Engineering Research and Post-Graduate Center, Mogi das Cruzes University (UMC), Mogi das Cruzes, SP, Brazil. ### Abstract Osteoarthritis (OA) triggers increased levels of inflammatory markers, including prostaglandin (PG) E2 and proinflammatory cytokines. The elevation of cytokine levels is closely associated with increased articular tissue degeneration. Thus, the use of combination therapies may presumably be able to enhance the effects on the modulation of inflammatory markers. The present study aimed to evaluate and compare the effects of photobiomodulation therapy (PBMT), physical exercise, and topical nonsteroidal anti-inflammatory drug (NSAID) use on the inflammatory process after they were applied either alone or in different combinations. OA was induced by intra-articular papain injection in the knee of rats. After 21 days, the animals began treatment with a topical NSAID and/or with physical exercise and/or PBMT. Treatments were performed three times a week for eight consecutive weeks, totaling 24 therapy sessions. Analysis of real-time polymerase chain reaction (RT-PCR) gene expression; interleukin (IL)-1?, IL-6, and tumor necrosis factor alpha (TNF-?) protein expression; and PGE2 levels by enzyme-linked immunosorbent assay (ELISA) was conducted. Our results showed that PBMT alone and Exerc + PBMT significantly reduced IL-1? gene expression (p?<?0.05) while no treatment changed both IL-6 and TNF-? gene expression. Treatment with NSAID alone, PBMT alone, Exerc + PBMT, and NSAID + PBMT reduced IL-1? protein expression (p<0.05). All therapies significantly reduced IL-6 and TNF-? protein expression (p<0.05) compared with the OA group. Similarly, all therapies, except Exerc, reduced the levels of PGE2 (p?<0.05) compared with the OA group. The results from the present study indicate that treatment with PBMT is more effective in modulating the inflammatory process underlying OA when compared with the other therapies tested. Lasers Med Sci. 2014 Mar;29(2):653-8. doi: 10.1007/s10103-013-1377-3. Epub 2013 Jun 30. # What is the best treatment to decrease pro-inflammatory cytokine release in acute skeletal muscle injury induced by trauma in rats: low-level laser therapy, diclofenac, or cryotherapy? de Almeida P1, Tomazoni SS, Frigo L, de Carvalho Pde T, Vanin AA, Santos LA, Albuquerque-Pontes GM, De Marchi T, Tairova O, Marcos RL, Lopes-Martins RÁ, Leal-Junior EC. ### Author information 1 Postgraduate Program in Rehabilitation Sciences, Universidade Nove de Julho (UNINOVE), São Paulo, SP, Brazil. ### Abstract Currently, treatment of muscle injuries represents a challenge in clinical practice. In acute phase, the most employed therapies are cryotherapy and nonsteroidal anti-inflammatory drugs. In the last years, low-level laser therapy (LLLT) has becoming a promising therapeutic agent; however, its effects are not fully known. The aim of this study was to analyze the effects of sodium diclofenac (topical application), cryotherapy, and LLLT on pro-inflammatory cytokine levels after a controlled model of muscle injury. For such, we performed a single trauma in tibialis anterior muscle of rats. After 1 h, animals were treated with sodium diclofenac (11.6 mg/g of solution), cryotherapy (20 min), or LLLT (904 nm; superpulsed; 700 Hz; 60 mW mean output power; 1.67 W/cm(2); 1, 3, 6 or 9 J; 17, 50, 100 or 150 s). Assessment of interleukin-1? and interleukin-6 (IL-1? and IL-6) and tumor necrosis factor-alpha (TNF-?) levels was performed at 6 h after trauma employing enzyme-linked immunosorbent assay method. LLLT with 1 J dose significantly decreased (p?<?0.05) IL-1?, IL-6, and TNF-? levels compared to non-treated injured group as well as diclofenac and cryotherapy groups. On the other hand, treatment with diclofenac and cryotherapy does not decrease pro-inflammatory cytokine levels compared to the non-treated injured group. Therefore, we can conclude that 904 nm LLLT with 1 J dose has better effects than topical application of diclofenac or cryotherapy in acute inflammatory phase after muscle trauma. Angle Orthod. 2010 Sep;80(5):925-32. doi: 10.2319/010410-10.1. # Interventions for pain during fixed orthodontic appliance therapy. A systematic review. Xiaoting L1, Yin T, Yangxi C. ### Author information 1 State Key Laboratory of Oral Disease and Department of Orthodontics, West China School of Dentistry, Sichuan University, Chengdu, China. ### Abstract #### OBJECTIVE: To compare the different methods of pain control intervention during fixed orthodontic appliance therapy. #### MATERIALS AND METHODS: A computerized literature search was performed in MEDLINE (1966-2009), The Cochrane Library (Issue 4, 2009), EMBASE (1984-2009), and CNKI (1994-2009) to collect randomized controlled trials (RCTs) for pain reduction during orthodontic treatment. Data were independently extracted by two reviewers and a quality assessment was carried out. The Cochrane Collaboration’s RevMan5 software was used for data analysis. The Cochrane Oral Health Group’s statistical guidelines were followed. #### RESULTS: Twenty-six RCTs were identified and six trials including 388 subjects were included. Meta-analysis showed that ibuprofen had a pain control effect at 6 hours and at 24 hours after archwire placement compared with the placebo group. The standard mean difference was -0.47 and -0.48, respectively. There was no difference in pain control between ibuprofen, acetaminophen, and aspirin. Other analgesics such as tenoxicam and valdecoxib had relatively lower visual analog scale (VAS) scores in pain perception. Lowlevel laser therapy (LLLT) was also an effective approach for pain relief with VAS scores of 3.30 in the LLLT group and 7.25 in the control group. #### CONCLUSIONS: Analgesics are still the main treatment modality to reduce orthodontic pain despite their side effects. Some long-acting nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase enzyme (COX-2) inhibitors are recommended for their comparatively lesser side effects. Their preemptive use is promising. Other approaches such as LLLT have aroused researchers’ attention. Photomed Laser Surg. 2010 Aug;28(4):553-60. doi: 10.1089/pho.2009.2576. # Acute low back pain with radiculopathy: a double-blind, randomized, placebo-controlled study. Konstantinovic LM1, Kanjuh ZM, Milovanovic AN, Cutovic MR, Djurovic AG, Savic VG, Dragin AS, Milovanovic ND. ### Author information ### Abstract #### OBJECTIVE: The aim of this study was to investigate the clinical effects of lowlevel laser therapy (LLLT) in patients with acute low back pain (LBP) with radiculopathy. #### BACKGROUND DATA: Acute LBP with radiculopathy is associated with pain and disability and the important pathogenic role of inflammation. LLLT has shown significant anti-inflammatory effects in many studies. #### MATERIALS AND METHODS: A randomized, double-blind, placebo-controlled trial was performed on 546 patients. Group A (182 patients) was treated with nimesulide 200 mg/day and additionally with active LLLT; group B (182 patients) was treated only with nimesulide; and group C (182 patients) was treated with nimesulide and placebo LLLT. LLLT was applied behind the involved spine segment using a stationary skin-contact method. Patients were treated 5 times weekly, for a total of 15 treatments, with the following parameters: wavelength 904 nm; frequency 5000 Hz; 100-mW average diode power; power density of 20 mW/cm(2) and dose of 3 J/cm(2); treatment time 150 sec at whole doses of 12 J/cm(2). The outcomes were pain intensity measured with a visual analog scale (VAS); lumbar movement, with a modified Schober test; pain disability, with Oswestry disability score; and quality of life, with a 12-item short-form health survey questionnaire (SF-12). Subjects were evaluated before and after treatment. Statistical analyses were done with SPSS 11.5. #### RESULTS: Statistically significant differences were found in all outcomes measured (p < 0.001), but were larger in group A than in B (p < 0.0005) and C (p < 0.0005). The results in group C were better than in group B (p < 0.0005). #### CONCLUSIONS: The results of this study show better improvement in acute LBP treated with LLLT used as additional therapy. J Oral Rehabil. 2008 Dec;35(12):925-33. doi: 10.1111/j.1365-2842.2008.01891.x. # Low–levellaser therapy improves bone repair in rats treated with anti-inflammatory drugs. Ribeiro DA1, Matsumoto MA. ### Author information 1 Department of Biosciences, Federal University of Sao Paulo, UNIFESP, Santos, SP, Brazil. daribeiro@unifesp.br ### Abstract Nowadays, selective cyclooxygenase-2 non-steroidal anti-inflammatory drugs have been largely used in surgical practice for reducing oedema and pain. However, the association between these drugs and laser therapy is not known up to now. Herein, the aim of this study was to evaluate the action of anti-COX-2 selective drug (celecoxib) on bone repair associated with laser therapy. A total of 64 rats underwent surgical bone defects in their tibias, being randomly distributed into four groups: Group 1) negative control; Group 2) animals treated with celecoxib; Group 3) animals treated with lowlevel power laser and Group 4) animals treated with celecoxib and lowlevel power laser. The animals were killed after 48 h, 7, 14 and 21 days. The tibias were removed for morphological, morphometric and immunohistochemistry analysis for COX-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups at 14 days after surgery for Groups 3 and 4. COX-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in the laser-exposed groups. Taken together, such results suggest that lowlevel laser therapy is able to improve bone repair in the tibia of rats as a result of an up-regulation for cyclooxygenase-2 expression in bone cells. Clin Orthop Relat Res. 2008 Jul;466(7):1539-54. doi: 10.1007/s11999-008-0260-1. Epub 2008 Apr 30. # Treatment of tendinopathy: what works, what does not, and what is on the horizon. Andres BM1, Murrell GA. ### Author information 1 Orthopaedic Research Institute, St George Hospital, University of New South Wales, Level 2 Research and Education Building, 4-10 South Street, Kogarah, Sydney, NSW, 2217, Australia. bandres@yahoo.com ### Abstract Tendinopathy is a broad term encompassing painful conditions occurring in and around tendons in response to overuse. Recent basic science research suggests little or no inflammation is present in these conditions. Thus, traditional treatment modalities aimed at controlling inflammation such as corticosteroid injections and nonsteroidal antiinflammatory medications (NSAIDS) may not be the most effective options. We performed a systematic review of the literature to determine the best treatment options for tendinopathy. We evaluated the effectiveness of NSAIDS, corticosteroid injections, exercise-based physical therapy, physical therapy modalities, shock wave therapy, sclerotherapy, nitric oxide patches, surgery, growth factors, and stem cell treatment. NSAIDS and corticosteroids appear to provide pain relief in the short term, but their effectiveness in the long term has not been demonstrated. We identified inconsistent results with shock wave therapy and physical therapy modalities such as ultrasound, iontophoresis and lowlevel laser therapy. Current data support the use of eccentric strengthening protocols, sclerotherapy, and nitric oxide patches, but larger, multicenter trials are needed to confirm the early results with these treatments. Preliminary work with growth factors and stem cells is promising, but further study is required in these fields. Surgery remains the last option due to the morbidity and inconsistent outcomes. The ideal treatment for tendinopathy remains unclear. ## Cognitive Enhancement Lasers Med Sci. 2017 May 2. doi: 10.1007/s10103-017-2221-y. [Epub ahead of print] # Beneficial neurocognitive effects of transcranial laser in older adults. Vargas E1, Barrett DW1, Saucedo CL1, Huang LD2, Abraham JA2, Tanaka H3, Haley AP1, Gonzalez-Lima F4. ### Author information 1 Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, TX, 78712, USA. 2 Department of Electrical Engineering, University of Texas at Austin, Austin, TX, 78712, USA. 3 Department of Kinesiology and Health Education, University of Texas at Austin, Austin, TX, 78712, USA. 4 Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, TX, 78712, USA. gonzalezlima@utexas.edu. ### Abstract Transcranial infrared laser stimulation (TILS) at 1064 nm, 250 mW/cm2 has been proven safe and effective for increasing neurocognitive functions in young adults in controlled studies using photobiomodulation of the right prefrontal cortex. The objective of this pilot study was to determine whether there is any effect from TILS on neurocognitive function in older adults with subjective memory complaint at risk for cognitive decline (e.g., increased carotid artery intima-media thickness or mild traumatic brain injury). We investigated the cognitive effects of TILS in older adults (ages 49-90, n = 12) using prefrontal cortex measures of attention (psychomotor vigilance task (PVT)) and memory (delayed match to sample (DMS)), carotid artery intima-media thickness (measured by ultrasound), and evaluated the potential neural mechanisms mediating the cognitive effects of TILS using exploratory brain studies of electroencephalography (EEG, n = 6) and functional magnetic resonance imaging (fMRI, n = 6). Cognitive performance, age, and carotid artery intima-media thickness were highly correlated, but all participants improved in all cognitive measures after TILS treatments. Baseline vs. chronic (five weekly sessions, 8 min each) comparisons of mean cognitive scores all showed improvements, significant for PVT reaction time (p < 0.001), PVT lapses (p < 0.001), and DMS correct responses (p < 0.05). The neural studies also showed for the first time that TILS increases resting-state EEG alpha, beta, and gamma power and promotes more efficient prefrontal blood-oxygen-level-dependent (BOLD)-fMRI response. Importantly, no adverse effects were found. These preliminary findings support the use of TILS for larger randomized clinical trials with this non-invasive approach to augment neurocognitive function in older people to combat aging-related and vascular disease-related cognitive decline. BBA Clin. 2016 Dec; 6: 113–124. Published online 2016 Oct 1. PMCID: PMC5066074 # Shining light on the head: Photobiomodulation for brain disorders Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA Michael R. Hamblin: ude.dravrah.hgm.xileh@nilbmaH ?Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA.Wellman Center for PhotomedicineMassachusetts General HospitalBostonMA02114USA ude.dravrah.hgm.xileh@nilbmaH Author information ? Article notes ? Copyright and License information ? Received 2016 Sep 2; Revised 2016 Sep 27; Accepted 2016 Sep 29. ## Abstract Photobiomodulation (PBM) describes the use of red or near-infrared light to stimulate, heal, regenerate, and protect tissue that has either been injured, is degenerating, or else is at risk of dying. One of the organ systems of the human body that is most necessary to life, and whose optimum functioning is most worried about by humankind in general, is the brain. The brain suffers from many different disorders that can be classified into three broad groupings: traumatic events (stroke, traumatic brain injury, and global ischemia), degenerative diseases (dementia, Alzheimer’s and Parkinson’s), and psychiatric disorders (depression, anxiety, post traumatic stress disorder). There is some evidence that all these seemingly diverse conditions can be beneficially affected by applying light to the head. There is even the possibility that PBM could be used for cognitive enhancement in normal healthy people. In this transcranial PBM (tPBM) application, near-infrared (NIR) light is often applied to the forehead because of the better penetration (no hair, longer wavelength). Some workers have used lasers, but recently the introduction of inexpensive light emitting diode (LED) arrays has allowed the development of light emitting helmets or “brain caps”. This review will cover the mechanisms of action of photobiomodulation to the brain, and summarize some of the key pre-clinical studies and clinical trials that have been undertaken for diverse brain disorders. Keywords: Photobiomodulation, Low level laser (light) therapy, Ischemic stroke, Traumatic brain injury, Alzheimer’s disease, Parkinson’s disease, Major depression, Cognitive enhancement ## Graphical abstract ## 1.?Introduction Photobiomodulation (PBM) as it is known today (the beneficial health benefits of light therapy had been known for some time before), was accidently discovered in 1967, when Endre Mester from Hungary attempted to repeat an experiment recently published by McGuff in Boston, USA [1]. McGuff had used a beam from the recently discovered ruby laser [2], to destroy a cancerous tumor that had been experimentally implanted into a laboratory rat. However (unbeknownst to Mester) the ruby laser that had been built for him, was only a tiny fraction of the power of the laser that had previously been used by McGuff. However, instead of curing the experimental tumors with his low-powered laser, Mester succeeded in stimulating hair regrowth and wound healing in the rats, in the sites where the tumors had been implanted [3], [4]. This discovery led to a series of papers describing what Mester called “laser biostimulation”, and soon became known as “low level laser therapy” (LLLT) [5], [6], [7]. LLLT was initially primarily studied for stimulation of wound healing, and reduction of pain and inflammation in various orthopedic conditions such as tendonitis, neck pain, and carpal tunnel syndrome [8]. The advent of light emitting diodes (LED) led to LLLT being renamed as “low level light therapy”, as it became more accepted that the use of coherent lasers was not absolutely necessary, and a second renaming occurred recently [9] when the term PBM was adopted due to uncertainties in the exact meaning of “low level”. ## 2.?Mechanisms of action of photobiomodulation ### 2.1. Mitochondria and cytochrome c oxidase The most well studied mechanism of action of PBM centers around cytochrome c oxidase (CCO), which is unit four of the mitochondrial respiratory chain, responsible for the final reduction of oxygen to water using the electrons generated from glucose metabolism [10]. The theory is that CCO enzyme activity may be inhibited by nitric oxide (NO) (especially in hypoxic or damaged cells). This inhibitory NO can be dissociated by photons of light that are absorbed by CCO (which contains two heme and two copper centers with different absorption spectra) [11]. These absorption peaks are mainly in the red (600–700 nm) and near-infrared (760–940 nm) spectral regions. When NO is dissociated, the mitochondrial membrane potential is increased, more oxygen is consumed, more glucose is metabolized and more ATP is produced by the mitochondria. ### 2.2. Reactive oxygen species, nitric oxide, blood flow It has been shown that there is a brief increase in reactive oxygen species (ROS) produced in the mitochondria when they absorb the photons delivered during PBM. The idea is that this burst of ROS may trigger some mitochondrial signaling pathways leading to cytoprotective, anti-oxidant and anti-apoptotic effects in the cells [12]. The NO that is released by photodissociation acts as a vasodilator as well as a dilator of lymphatic flow. Moreover NO is also a potent signaling molecule and can activate a number of beneficial cellular pathways [13]. Fig. 2 illustrates these mechanisms. Tissue specific processes that occur after PBM and benefit a range of brain disorders. BDNF, brain-derived neurotrophic factor; LLLT, low level light therapy; NGF, nerve growth factor; NT-3, neurotrophin 3; PBM, photobiomodulation; SOD, superoxide dismutase. ### 2.3. Light sensitive ion channels and calcium It is quite clear that there must be some other type of photoacceptor, in addition to CCO, as is clearly demonstrated by the fact that wavelengths substantially longer than the red/NIR wavelengths discussed above, can also produce beneficial effects is some biological scenarios. Wavelengths such as 980 nm [14], [15], 1064 nm laser [16], and 1072 nm LED [17], and even broad band IR light [18] have all been reported to carry out PBM type effects. Although the photoacceptor for these wavelengths has by no means been conclusively identified, the leading hypothesis is that it is primarily water (perhaps nanostructured water) located in heat or light sensitive ion channels. Clear changes in intracellular calcium can be observed, that could be explained by light-mediated opening of calcium ion channels, such as members of the transient receptor potential (TRP) super-family [19]. TRP describes a large family of ion channels typified by TRPV1, recently identified as the biological receptor for capsaicin (the active ingredient in hot chili peppers) [20]. The biological roles of TRP channels are multifarious, but many TRP channels are involved in heat sensing and thermoregulation [21]. ### 2.4. Signaling mediators and activation of transcription factors Most authors suggest that the beneficial effects of tPBM on the brain can be explained by increases in cerebral blood flow, greater oxygen availability and oxygen consumption, improved ATP production and mitochondrial activity [22], [23], [24]. However there are many reports that a brief exposure to light (especially in the case of experimental animals that have suffered some kind of acute injury or traumatic insult) can have effects lasting days, weeks or even months [25]. This long-lasting effect of light can only be explained by activation of signaling pathways and transcription factors that cause changes in protein expression that last for some considerable time. The effects of PBM on stimulating mitochondrial activity and blood flow is of itself, unlikely to explain long-lasting effects. A recent review listed no less than fourteen different transcription factors and signaling mediators, that have been reported to be activated after light exposure [10]. Fig. 1 illustrates two of the most important molecular photoreceptors or chromophores (cytochrome c oxidase and heat-gated ion channels) inside neuronal cells that absorb photons that penetrate into the brain. The signaling pathways and activation of transcription factors lead to the eventual effects of PBM in the brain. Molecular and intracellular mechanisms of transcranial low level laser (light) or photobiomodulation. AP1, activator protein 1; ATP, adenosine triphosphate; Ca2 +, calcium ions; cAMP, cyclic adenosine monophosphate; NF-kB, nuclear factor kappa Fig. 2 illustrates some more tissue specific mechanisms that lead on from the initial photon absorption effects explained in Fig. 1. A wide variety of processes can occur that can benefit a correspondingly wide range of brain disorders. These processes can be divided into short-term stimulation (ATP, blood flow, lymphatic flow, cerebral oxygenation, less edema). Another group of processes center around neuroprotection (upregulation of anti-apoptotic proteins, less excitotoxity, more antioxidants, less inflammation). Finally a group of processes that can be grouped under “help the brain to repair itself” (neurotrophins, neurogenesis and synaptogenesis). ### 2.5. Biphasic dose response and effect of coherence The biphasic dose response (otherwise known as hormesis, and reviewed extensively by Calabrese et al. [26]) is a fundamental biological law describing how different biological systems can be activated or stimulated by low doses of any physical insult or chemical substance, no matter how toxic or damaging this insult may be in large doses. The most well studied example of hormesis is that of ionizing radiation, where protective mechanisms are induced by very low exposures, that can not only protect against subsequent large doses of ionizing radiation, but can even have beneficial effects against diseases such as cancer using whole body irradiation [27]. There are many reports of PBM following a biphasic dose response (sometimes called obeying the Arndt-Schulz curve [28], [29]. A low dose of light is beneficial, but raising the dose produces progressively less benefit until eventually a damaging effect can be produced at very high light [30]. It is often said in this context that “more does not mean more”. Another question that arises in the field of PBM is whether the coherent monochromatic lasers that were used in the original discovery of the effect, and whose use continued for many years, are superior to the rather recent introduction of LEDs, that are non-coherent and have a wider band-spread (generally 30 nm full-width half-maximum). Although there are one or two authors who continue to believe that coherent lasers are superior [31], most commentators feel that other parameters such as wavelength, power density, energy density and total energy are the most important determinants of efficacy [8]. ## 3.?Tissue optics, direct versus systemic effects, light sources ### 3.1. Light penetration into the brain Due to the growing interest in PBM of the brain, several tissue optics laboratories have investigated the penetration of light of different wavelengths through the scalp and the skull, and to what depths into the brain this light can penetrate. This is an intriguing question to consider, because at present it is unclear exactly what threshold of power density in mW/cm2 is required in the b5rain to have a biological effect. There clearly must be a minimum value below which the light can be delivered for an infinite time without doing anything, but whether this is in the region of ?W/cm2 or mW/cm2 is unknown at present. Functional near-infrared spectroscopy (fNIRS) using 700–900 nm light has been established as a brain imaging technique that can be compared to functional magnetic resonance imaging (fMRI) [32]. Haeussinger et al. estimated that the mean penetration depth (5% remaining intensity) of NIR light through the scalp and skull was 23:6 + 0:7 mm [33]. Other studies have found comparable results with variations depending on the precise location on the head and wavelength [34], [35]. Jagdeo et al. [36] used human cadaver heads (skull with intact soft tissue) to measure penetration of 830 nm light, and found that penetration depended on the anatomical region of the skull (0.9% at the temporal region, 2.1% at the frontal region, and 11.7% at the occipital region). Red light (633 nm) hardly penetrated at all. Tedord et al. [37] also used human cadaver heads to compare penetration of 660 nm, 808 nm, and 940 nm light. They found that 808 nm light was best and could reach a depth in the brain of 40–50 mm. Lapchak et al. compared the transmission of 810 nm light through the skulls of four different species, and found mouse transmitted 40%, while for rat it was 21%, rabbit it was 11.3 and for human skulls it was only 4.2% [38]. Pitzschke and colleagues compared penetration of 670 nm and 810 nm light into the brain when delivered by a transcranial or a transphenoidal approach, and found that the best combination was 810 nm delivered transphenoidally [39]. In a subsequent study these authors compared the effects of storage and processing (frozen or formalin-fixed) on the tissue optical properties of rabbit heads [40]. Yaroslavsky et al. examined light penetration of different wavelengths through different parts of the brain tissue (white brain matter, gray brain matter, cerebellum, and brainstem tissues, pons, thalamus). Best penetration was found with wavelengths between 1000 and 1100 nm [41]. Henderson and Morries found that between 0.45% and 2.90% of 810 nm or 980 nm light penetrated through 3 cm of scalp, skull and brain tissue in ex vivo lamb heads [42]. ### 3.2. Systemic effects It is in fact very likely that the beneficial effects of PBM on the brain cannot be entirely explained by penetration of photons through the scalp and skull into the brain itself. There have been some studies that have explicitly addressed this exact issue. In a study of PBM for Parkinson’s disease in a mouse model [43]. Mitrofanis and colleagues compared delivering light to the mouse head, and also covered up the head with aluminum foil so that they delivered light to the remainder of the mouse body. They found that there was a highly beneficial effect on neurocognitive behavior with irradiation to the head, but nevertheless there was also a statistically significant (although less pronounced benefit, referred to by these authors as an ‘abscopal effect”) when the head was shielded from light [44]. Moreover Oron and co-workers [45] have shown that delivering NIR light to the mouse tibia (using either surface illumination or a fiber optic) resulted in improvement in a transgenic mouse model of Alzheimer’s disease (AD). Light was delivered weekly for 2 months, starting at 4 months of age (progressive stage of AD). They showed improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. They proposed that the mechanism of this effect was to stimulate c-kit-positive mesenchymal stem cells (MSCs) in autologous bone marrow (BM) to enhance the capacity of MSCs to infiltrate the brain, and clear ?-amyloid plaques [46]. It should be noted that the calvarial bone marrow of the skull contains substantial numbers of stem cells [47]. ### 3.3. Laser acupuncture Laser acupuncture is often used as an alternative or as an addition to traditional Chinese acupuncture using needles [48]. Many of the applications of laser acupuncture have been for conditions that affect the brain [49] such as Alzheimer’s disease [50] and autism [51] that have all been investigated in animal models. Moreover laser acupuncture has been tested clinically [52]. ### 3.4. Light sources A wide array of different light sources (lasers and LEDs) have been employed for tPBM. One of the most controversial questions which remains to be conclusively settled, is whether a coherent monochromatic laser is superior to non-coherent LEDs typically having a 30 nm band-pass (full width half maximum). Although wavelengths in the NIR region (800–1100 nm) have been the most often used, red wavelengths have sometimes been used either alone, or in combination with NIR. Power levels have also varied markedly from Class IV lasers with total power outputs in the region of 10 W [53], to lasers with more modest power levels (circa 1 W). LEDs can also have widely varying total power levels depending on the size of the array and the number and power of the individual diodes. Power densities can also vary quite substantially from the Photothera laser [54] and other class IV lasers , which required active cooling (~ 700 mW/cm2) to LEDs in the region of 10–30 mW/cm2. ### 3.5. Usefulness of animal models when testing tPBM for brain disorders One question that is always asked in biomedical research, is how closely do the laboratory models of disease (which are usually mice or rats) mimic the human disease for which new treatments are being sought? This is no less critical a question when the areas being studied include brain disorders and neurology. There now exist a plethora of transgenic mouse models of neurological disease [55], [56]. However in the present case, where the proposed treatment is almost completely free of any safety concerns, or any reported adverse side effects, it can be validly questioned as to why the use of laboratory animal models should be encouraged. Animal models undoubtedly have disadvantages such as failure to replicate all the biological pathways found in human disease, difficulty in accurately measuring varied forms of cognitive performance, small size of mice and rats compared to humans, short lifespan affecting the development of age related diseases, and lack of lifestyle factors that adversely affect human diseases. Nevertheless, small animal models are less expensive, and require much less time and effort to obtain results than human clinical trials, so it is likely they will continue to be used to test tPBM for the foreseeable future. ## 4.?PBM for stroke ### 4.1. Animal models Perhaps the most well-investigated application of PBM to the brain, lies in its possible use as a treatment for acute stroke [57]. Animal models such as rats and rabbits, were first used as laboratory models, and these animals had experimental strokes induced by a variety of methods and were then treated with light (usually 810 nm laser) within 24 h of stroke onset [58]. In these studies intervention by tLLLT within 24 h had meaningful beneficial effects. For the rat models, stroke was induced by middle cerebral artery occlusion (MCAO) via an insertion of a filament into the carotid artery or via craniotomy [59], [60]. Stroke induction in the “rabbit small clot embolic model” (RSCEM) was by injection of a preparation of small blood clots (made from blood taken from a second donor rabbit) into a catheter placed in the right internal carotid artery [61]. These studies and the treatments and results are listed in Table 1. Reports of transcranial LLLT used for stroke in animal models. CW, continuous wave; LLLT, low level light therapy; MCAO, middle cerebral artery occlusion; NOS, nitric oxide synthase; RSCEM, rabbit small clot embolic model; TGF?1, transforming growth factor ?1. ### 4.2. Clinical trials for acute stroke Treatment of acute stroke was addressed in a series of three clinical trials called “Neurothera Effectiveness and Safety Trials” (NEST-1 [65], NEST-2 [66], and NEST-3 [67]) using an 810 nm laser applied to the shaved head within 24 h of patients suffering an ischemic stroke. The first study, NEST-1, enrolled 120 patients between the ages of 40 to 85 years of age with a diagnosis of ischemic stroke involving a neurological deficit that could be measured. The purpose of this first clinical trial was to demonstrate the safety and effectiveness of laser therapy for stroke within 24 h [65]. tPBM significantly improved outcome in human stroke patients, when applied at ~ 18 h post-stroke, over the entire surface of the head (20 points in the 10/20 EEG system) regardless of stroke [65]. Only one laser treatment was administered, and 5 days later, there was significantly greater improvement in the Real- but not in the Sham-treated group (p < 0.05, NIH Stroke Severity Scale). This significantly greater improvement was still present at 90 days post-stroke, where 70% of the patients treated with Real-LLLT had a successful outcome, while only 51% of Sham-controls did. The second clinical trial, NEST-2, enrolled 660 patients, aged 40 to 90, who were randomly assigned to one of two groups (331 to LLLT, 327 to sham) [68]. Beneficial results (p < 0.04) were found for the moderate and moderate-severe (but not for the severe) stroke patients, who received the Real laser protocol [68]. These results suggested that the overall severity of the individual stroke should be taken into consideration in future studies, and very severe patients are unlikely to recover with any kind of treatment. The last clinical trial, NEST-3, was planned for 1000 patients enrolled. Patients in this study were not to receive tissue plasminogen activator, but the study was prematurely terminated by the DSMB for futility (an expected lack of statistical significance) [67]. NEST-1 was considered successful, even though as a phase 1 trial, it was not designed to show efficacy. NEST-2 was partially successful when the patients were stratified, to exclude very severe strokes or strokes deep within the brain [66]. There has been considerable discussion in the scientific literature on precisely why the NEST-3 trial failed [69]. Many commentators have wondered how could tPBM work so well in the first trial, in a sub-group in the second trial, and fail in the third trial. Lapchak’s opinion is that the much thicker skull of humans compared to that of the other animals discussed above (mouse, rat and rabbit), meant that therapeutically effective amounts of light were unlikely to reach the brain [69]. Moreover the time between the occurrence of a stroke and initiation of the PBMT may be an important factor. There are reports in the literature that neuroprotection must be administered as soon as possible after a stroke [70], [71]. Furthermore, stroke trials in particular should adhere to the RIGOR (rigorous research) guidelines and STAIR (stroke therapy academic industry roundtable) criteria [72]. Other contributory causes to the failure of NEST-3 may have been included the decision to use only one single tPBM treatment, instead of a series of treatments. Moreover, the optimum brain areas to be treated in acute stroke remain to be determined. It is possible that certain areas of the brain that have sustained ischemic damage should be preferentially illuminated and not others. ### 4.3. Chronic stroke Somewhat surprisingly, there have not as yet been many trials of PBM for rehabilitation of stroke patients with only the occasional report to date. Naeser reported in an abstract the use of tPBM to treat chronic aphasia in post-stroke patients [73]. Boonswang et al. [74] reported a single patient case in which PBM was used in conjunction with physical therapy to rehabilitate chronic stroke damage. However the findings that PBM can stimulate synaptogenesis in mice with TBI, does suggest that tPBM may have particular benefits in rehabilitation of stroke patients. Norman Doidge, in Toronto, Canada has described the use of PBM as a component of a neuroplasticity approach to rehabilitate chronic stroke patients [75]. ## 5. PBM for traumatic brain injury (TBI) ### 5.1. Mouse and rat models There have been a number of studies looking at the effects of PBM in animal models of TBI. Oron’s group was the first [76] to demonstrate that a single exposure of the mouse head to a NIR laser (808 nm) a few hours after creation of a TBI lesion could improve neurological performance and reduce the size of the brain lesion. A weight-drop device was used to induce a closed-head injury in the mice. An 808 nm diode laser with two energy densities (1.2–2.4 J/cm2 over 2 min of irradiation with 10 and 20 mW/cm2) was delivered to the head 4 h after TBI was induced. Neurobehavioral function was assessed by the neurological severity score (NSS). There were no significant difference in NSS between the power densities (10 vs 20 mW/cm2) or significant differentiation between the control and laser treated group at early time points (24 and 48 h) post TBI. However, there was a significant improvement (27% lower NSS score) in the PBM group at times of 5 days to 4 weeks. The laser treated group also showed a smaller loss of cortical tissue than the sham group [76]. Hamblin’s laboratory then went on (in a series of papers [76]) to show that 810 nm laser (and 660 nm laser) could benefit experimental TBI both in a closed head weight drop model [77], and also in controlled cortical impact model in mice [25]. Wu et al. [77] explored the effect that varying the laser wavelengths of LLLT had on closed-head TBI in mice. Mice were randomly assigned to LLLT treated group or to sham group as a control. Closed-head injury (CHI) was induced via a weight drop apparatus. To analyze the severity of the TBI, the neurological severity score (NSS) was measured and recorded. The injured mice were then treated with varying wavelengths of laser (665, 730, 810 or 980 nm) at an energy level of 36 J/cm2 at 4 h directed onto the scalp. The 665 nm and 810 nm groups showed significant improvement in NSS when compared to the control group at day 5 to day 28. Results are shown in Fig. 3. Conversely, the 730 and 980 nm groups did not show a significant improvement in NSS and these wavelengths did not produce similar beneficial effects as in the 665 nm and 810 nm LLLT groups [77]. The tissue chromophore cytochrome c oxidase (CCO) is proposed to be responsible for the underlying mechanism that produces the many PBM effects that are the byproduct of LLLT. COO has absorption bands around 665 nm and 810 nm while it has low absorption bands at the wavelength of 730 nm [78]. It should be noted that this particular study found that the 980 nm did not produce the same positive effects as the 665 nm and 810 nm wavelengths did; nevertheless previous studies did find that the 980 nm wavelength was an active one for LLLT. Wu et al. proposed that these dissimilar results may be due to the variance in the energy level, irradiance, etc. between the other studies and this particular study [77]. tPBM for TBI in a mouse model. Mice received a closed head injury and 4 hours later a single exposure of the head to one of four different lasers (36 J/cm2 delivered at 150 mW/cm2 over 4 min with spot size 1-cm diameter) Ando et al. [25] used the 810 nm wavelength laser parameters from the previous study and varied the pulse modes of the laser in a mouse model of TBI. These modes consisted of either pulsed wave at 10 Hz or at 100 Hz (50% duty cycle) or continuous wave laser. For the mice, TBI was induced with a controlled cortical impact device via open craniotomy. A single treatment with an 810 nm Ga-Al-As diode laser with a power density of 50 mW/m2 and an energy density of 36 J/cm2 was given via tLLLT to the closed head in mice for a duration of 12 min at 4 h post CCI. At 48 h to 28 days post TBI, all laser treated groups had significant decreases in the measured neurological severity score (NSS) when compared to the control (Fig. 4A). Although all laser treated groups had similar NSS improvement rates up to day 7, the PW 10 Hz group began to show greater improvement beyond this point as seen in Fig. 4. At day 28, the forced swim test for depression and anxiety was used and showed a significant decrease in the immobility time for the PW 10 Hz group. In the tail suspension test which measures depression and anxiety, there was also a significant decrease in the immobility time at day 28, and this time also at day 1, in the PW 10 Hz group. tPBM for controlled cortical impact TBI in a mouse model. (A) Mice received a single exposure (810 nm laser, 36 J/cm2 delivered at 50 mW/cm2 over 12 min) [121]. (B) Mice received 3 daily exposures starting 4 h post-TBI Studies using immunofluorescence of mouse brains showed that tPBM increased neuroprogenitor cells in the dentate gyrus (DG) and subventricular zone at 7 days after the treatment [79]. The neurotrophin called brain derived neurotrophic factor (BDNF) was also increased in the DG and SVZ at 7 days , while the marker (synapsin-1) for synaptogenesis and neuroplasticity was increased in the cortex at 28 days but not in the DG, SVZ or at 7 days [80] (Fig. 4B). Learning and memory as measured by the Morris water maze was also improved by tPBM [81]. Whalen’s laboratory [82] and Whelan’s laboratory [83] also successfully demonstrated therapeutic benefits of tPBM for TBI in mice and rats respectively. Zhang et al. [84] showed that secondary brain injury occurred to a worse degree in mice that had been genetically engineered to lack “Immediate Early Response” gene X-1 (IEX-1) when exposed to a gentle head impact (this injury is thought to closely resemble mild TBI in humans). Exposing IEX-1 knockout mice to LLLT 4 h post injury, suppressed proinflammatory cytokine expression of interleukin (IL)-I? and IL-6, but upregulated TNF-?. The lack of IEX-1 decreased ATP production, but exposing the injured brain to LLLT elevated ATP production back to near normal levels. Dong et al. [85] even further improved the beneficial effects of PBM on TBI in mice, by combining the treatment with metabolic substrates such as pyruvate and/or lactate. The goal was to even further improve mitochondrial function. This combinatorial treatment was able to reverse memory and learning deficits in TBI mice back to normal levels, as well as leaving the hippocampal region completely protected from tissue loss; a stark contrast to that found in control TBI mice that exhibited severe tissue loss from secondary brain injury. ### 5.2. TBI in humans Margaret Naeser and collaborators have tested PBM in human subjects who had suffered TBI in the past [86]. Many sufferers from severe or even moderate TBI, have very long lasting and even life-changing sequelae (headaches, cognitive impairment, and difficulty sleeping) that prevent them working or living any kind or normal life. These individuals may have been high achievers before the accident that caused damage to their brain [87]. Initially Naeser published a report [88] describing two cases she treated with PBM applied to the forehead twice a week. A 500 mW continuous wave LED source (mixture of 660 nm red and 830 nm NIR LEDs) with a power density of 22.2 mW/cm2 (area of 22.48 cm2), was applied to the forehead for a typical duration of 10 min (13.3 J/cm2). In the first case study the patient reported that she could concentrate on tasks for a longer period of time (the time able to work at a computer increased from 30 min to 3 h). She had a better ability to remember what she read, decreased sensitivity when receiving haircuts in the spots where LLLT was applied, and improved mathematical skills after undergoing LLLT. The second patient had statistically significant improvements compared to prior neuropsychological tests after 9 months of treatment. The patient had a 2 standard deviation (SD) increase on tests of inhibition and inhibition accuracy (9th percentile to 63rd percentile on the Stroop test for executive function and a 1 SD increase on the Wechsler Memory scale test for the logical memory test (83rd percentile to 99th percentile) [89]. Naeser et al. then went on to report a case series of a further eleven patients [90]. This was an open protocol study that examined whether scalp application of red and near infrared (NIR) light could improve cognition in patients with chronic, mild traumatic brain injury (mTBI). This study had 11 participants ranging in age from 26 to 62 (6 males, 5 females) who suffered from persistent cognitive dysfunction after mTBI. The participants’ injuries were caused by motor vehicle accidents, sports related events and for one participant, an improvised explosive device (IED) blast. tLLLT consisted of 18 sessions (Monday, Wednesday, and Friday for 6 weeks) and commenced anywhere from 10 months to 8 years post-TBI. A total of 11 LED clusters (5.25 cm in diameter, 500 mW, 22.2 mW/cm2, 13 J/cm2) were applied for about 10 min per session (5 or 6 LED placements per set, Set A and then Set B, in each session). Neuropsychological testing was performed pre-LED application and 1 week, 1 month and 2 months after the final treatment. Naeser and colleagues found that there was a significant positive linear trend observed for the Stroop Test for executive function, in trial 2 inhibition (p = 0.004); Stroop, trial 4 inhibition switching (p = 0.003); California Verbal Learning Test (CVLT)-II, total trials 1–5 (p = 0.003); CVLT-II, long delay free recall (p = 0.006). Improved sleep and fewer post-traumatic stress disorder (PTSD) symptoms, if present beforehand, were observed after treatment. Participants and family members also reported better social function and a better ability to perform interpersonal and occupational activities. Although these results were significant, further placebo-controlled studies will be needed to ensure the reliability of this these data [90]. Henderson and Morries [91] used a high-power NIR laser (10–15 W at 810 and 980 nm) applied to the head to treat a patient with moderate TBI. The patient received 20 NIR applications over a 2-month period. They carried out anatomical magnetic resonance imaging (MRI) and perfusion single-photon emission computed tomography (SPECT). The patient showed decreased depression, anxiety, headache, and insomnia, whereas cognition and quality of life improved, accompanied by changes in the SPECT imaging. ## 6. PBM for Alzheimer’s disease (AD) ### 6.1. Animal models There was a convincing study [92] carried out in an A?PP transgenic mouse of AD. tPBM (810 nm laser) was administered at different doses 3 times/week for 6 months starting at 3 months of age. The numbers of A? plaques were significantly reduced in the brain with administration of tPBM in a dose-dependent fashion. tPBM mitigated the behavioral effects seen with advanced amyloid deposition and reduced the expression of inflammatory markers in the transgenic mice. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos expression suggesting that there was an overall improvement in neurological function. ### 6.2. Humans There has been a group of investigators in Northern England who have used a helmet built with 1072 nm LEDs to treat AD, but somewhat surprisingly no peer-reviewed publications have described this approach [93]. However a small pilot study (19 patients) that took the form of a randomized placebo-controlled trial investigated the effect of the Vielight Neuro system (see Fig. 5A) (a combination of tPBM and intranasal PBM) on patients with dementia and mild cognitive impairment [94]. This was a controlled single blind pilot study in humans to investigate the effects of PBM on memory and cognition. 19 participants with impaired memory/cognition were randomized into active and sham treatments over 12 weeks with a 4-week no-treatment follow-up period. They were assessed with MMSE and ADAS-cog scales. The protocol involved in-clinic use of a combined transcranial-intranasal PBM device; and at-home use of an intranasal-only PBM device and participants/ caregivers noted daily experiences in a journal. Active participants with moderate to severe impairment (MMSE scores 5–24) showed significant improvements (5-points MMSE score) after 12 weeks. There was also a significant improvement in ADAS-cog scores (see Fig. 5B). They also reported better sleep, fewer angry outbursts and decreased anxiety and wandering. Declines were noted during the 4-week no-treatment follow-up period. Participants with mild impairment to normal (MMSE scores of 25 to 30) in both the active and sham sub-groups showed improvements. No related adverse events were reported. tPBM for Alzheimer’s disease. (A) Nineteen patients were randomized to receive real or sham tPBM (810 nm LED, 24.6 J/cm2 at 41 mW/cm2). (B) Significant decline in ADAS-cog (improved cognitive performance) in real but not sham (unpublished An interesting paper from Russia [95] described the use of intravascular PBM to treat 89 patients with AD who received PBM (46 patients) or standard treatment with memantine and rivastigmine (43 patients). The PBM consisted of threading a fiber-optic through a cathéter in the fémoral artery and advancing it to the distal site of the anterior and middle cerebral arteries and delivering 20 mW of red laser for 20–40 min. The PBM group had improvement in cerebral microcirculation leading to permanent (from 1 to 7 years) reduction in dementia and cognitive recovery. ## 7. Parkinson’s disease The majority of studies on PBM for Parkinson’s disease have been in animal models and have come from the laboratory of John Mitrofanis in Australia [96]. Two basic models of Parkinson’s disease were used. The first employed administration of the small molecule (MPTP or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to mice [97]. MPTP was discovered as an impurity in an illegal recreational drug to cause Parkinson’s like symptoms (loss of substantia nigra cells) in young people who had taken this drug [98]. Mice were treated with tPBM (670-nm LED, 40 mW/cm2, 3.6 J/cm2) 15 min after each MPTP injection repeated 4 times over 30 h. There were significantly more (35%–45%) dopaminergic cells in the brains of the tPBM treated mice [97]. A subsequent study showed similar results in a chronic mouse model of MPTP-induced Parkinson’s disease [99]. They repeated their studies in another mouse model of Parkinson’s disease, the tau transgenic mouse strain (K3) that has a progressive degeneration of dopaminergic cells in the substantia nigra pars compacta (SNc) [100]. They went on to test a surgically implanted intracranial fiber designed to deliver either 670 nm LED (0.16 mW) or 670 nm laser (67 mW) into the lateral ventricle of the brain in MPTP-treated mice [101]. Both low power LED and high power laser were effective in preserving SNc cells, but the laser was considered to be unsuitable for long-term use (6 days) due to excessive heat production. As mentioned above, these authors also reported a protective effect of abscopal light exposure (head shielded) in this mouse model [43]. Recently this group has tested their implanted fiber approach in a model of Parkinson’s disease in adult Macaque monkeys treated with MPTP [102]. Clinical evaluation of Parkinson’s symptoms (posture, general activity, bradykinesia, and facial expression) in the monkeys were improved at low doses of light (24 J or 35 J) compared to high doses (125 J) [103]. The only clinical report of PBM for Parkinson’s disease in humans was an abstract presented in 2010 [104]. Eight patients between 18 and 80 years with late stage PD participated in a non-controlled, non-randomized study. Participants received tPBM treatments of the head designed to deliver light to the brain stem, bilateral occipital, parietal, temporal and frontal lobes, and treatment along the sagittal suture. A Visual Analog Scale (VAS), was used to record the severity of their symptoms of balance, gait, freezing, cognitive function, rolling in bed, and difficulties with speech pre-procedure and at study endpoint with 10 being most severe and 0 as no symptom. Compared with baseline, all participants demonstrated a numerical improvement in the VAS from baseline to study endpoint. A statistically significant reduction in VAS rating for gait and cognitive function was observed with average mean change of —1.87 (p < 0.05) for gait and a mean reduction of —2.22 (p < 0.05) for cognitive function. Further, freezing and difficulty with speech ratings were significantly lower (mean reduction of 1.28 (p < 0.05) for freezing and 2.22 (p < 0.05) for difficulty with speech). ## 8. PBM for psychiatric disorders ### 8.1. Animal models A common and well-accepted animal model of depression is called “chronic mild stress” [105]. After exposure to a series of chronic unpredictable mild stressors, animals develop symptoms seen in human depression, such as anhedonia (loss of the capacity to experience pleasure, a core symptom of major depressive disorder), weight loss or slower weight gain, decrease in locomotor activity, and sleep disorders [106]. Wu et al. used Wistar rats to show that after 5 weeks of chronic stress, application of tPBM 3 times a week for 3 weeks (810 nm laser, 100 Hz with 20% duty cycle, 120 J/cm2) gave significant improvement in the forced swimming test (FST) [107]. In a similar study Salehpour et al. [108] compared the effects of two different lasers (630 m nm at 89 mW/cm2, and 810 nm at 562 mW/cm2, both pulsed at 10 Hz, 50% duty cycle). The 810 nm laser proved better than the 630 nm laser in the FST, in the elevated plus maze and also reduced blood cortisol levels. ### 8.2. Depression and anxiety The first clinical study in depression and anxiety was published by Schiffer et al. in 2009 [109]. They used a fairly small area 1 W 810 nm LED array (see Fig. 6A) applied to the forehead in patients with major depression and anxiety. They found improvements in the Hamilton depression rating scale (HAM-D) (see Fig. 6B), and the Hamilton anxiety rating scale (HAM-A), 2 weeks after a single treatment. They also found increases in frontal pole regional cerebral blood flow (rCBF) during the light delivery using a commercial NIR spectroscopy device. Cassano and co-workers [110] used tPBM with an 810 nm laser (700 mW/cm2and a fluence of 84 J/cm2 delivered per session for 6 sessions in patients with major depression. Baseline mean HAM-D17 scores decreased from 19.8 ± 4.4 (SD) to 13 ± 5.35 (SD) after treatment (p = 0.004). tPBM for major depression and anxiety in humans. (A) Ten patients received a single exposure to the forehead (810 LED, 60 J/cm2delivered at 250 mW/cm2). (B) Mean Hamilton score for depression at baseline and at two weeks post-treatment ## 9. Cognitive enhancement From what we have seen above, it need come as no surprise, to learn that there are several reports about cognitive enhancement in normal people or healthy animals using PBM. The first report was in middle aged (12 months) CD1 female mice [111]. Exposure of the mice to 1072 nm LED arrays led to improved performance in a 3D maze compared to sham treated age-matched controls. Francisco Gonzalez-Lima at the University of Texas Austin, has worked in this area for some time [112]. Working in rats they showed that transcranial PBM (9 mW/cm2 with 660 nm LED array) induced a dose-dependent increase in oxygen consumption of 5% after 1 J/cm2 and 16% after 5 J/cm2 [113]. They also found that tPBM reduced fear renewal and prevented the reemergence of extinguished conditioned fear responses [113]. In normal human volunteers they used transcranial PBM (1064 nm laser, 60 J/cm2 at 250 mW/cm2) delivered to the forehead in a placebo-controlled, randomized study, to influence cognitive tasks related to the prefrontal cortex, including a psychomotor vigilance task (PVT), a delayed match-to-sample (DMS) memory task, and the positive and negative affect schedule (PANAS-X) to show improved mood [16]. Subsequent studies in normal humans showed that tPBM with 1064 nm laser could improve performance in the Wisconsin Card Sorting Task (considered the gold standard test for executive function) [114]. They also showed that tPBM to the right forehead (but not the left forehead) had better effects on improving attention bias modification (ABM) in humans with depression [115]. A study by Salgado et al. used transcranial LED PBM on cerebral blood flow in healthy elderly women analyzed by transcranial Doppler ultrasound (TCD) of the right and left middle cerebral artery and basilar artery. Twenty-five non-institutionalized elderly women (mean age 72 years old), with cognitive status > 24, were assessed using TCD before and after transcranial LED therapy. tPBM (627 nm, 70 mW/cm2, 10 J/cm2) was performed at four points of the frontal and parietal region for 30 s each twice a week for 4 weeks. There was a significant increase in the systolic and diastolic velocity of the left middle cerebral artery (25 and 30%, respectively) and the basilar artery (up to 17 and 25%), as well as a decrease in the pulsatility index and resistance index values of the three cerebral arteries analyzed [116]. ## 10. Conclusion Many investigators believe that PBM for brain disorders will become one of the most important medical applications of light therapy in the coming years and decades. Despite the efforts of “Big Pharma”, prescription drugs for psychiatric disorders are not generally regarded very highly (either by the medical profession or by the public), and many of these drugs perform little better than placebos in different trials, and moreover can also have major side-effects [117]. Moreover it is well accepted that with the overall aging of the general population, together with ever lengthening life spans, that dementia, Alzheimer’s, and Parkinson’s diseases will become a global health problem [118], [119]. Even after many years of research, no drug has yet been developed to benefit these neurodegenerative disorders. A similar state of play exists with drugs for stroke (with the exception of clot-busting enzymes) and TBI. New indications for tPBM such as global ischemia (brain damage after a heart attack), post-operative cognitive dysfunction [120], and neurodevelopmental disorders such as autism spectrum disorder may well emerge. Table 2 shows the wide range of brain disorders and diseases that may eventually be treated by some kind of tPBM, whether that be an office/clinic based procedure or a home-use based device. If inexpensive LED helmets can be developed and successfully marketed as home use devices, then we are potentially in a position to benefit large numbers of patients (to say nothing of healthy individuals). Certainly the advent of the Internet has made it much easier for knowledge about this kind of home treatment to spread (almost by word of mouth so to speak). List of brain disorders that may in principle be treated by tPBM. ## Conflict of interest statement The author declares no conflict of interest. ## Transparency document Transparency document. Click here to view.(1.1M, pdf)Image 1 ## Acknowledgments MRH was supported by the US NIH grants R01AI050875 and R21AI121700, the Air Force Office of Scientific Research grant FA9550-13-1-0068, the US Army Medical Research Acquisition Activity grant W81XWH-09-1-0514, and by the US Army Medical Research and Materiel Command grant W81XWH-13-2-0067. ## Footnotes The Transparency document associated with this article can be found, in online version. ## References 1. McGuff P.E., Deterling R.A., Jr., Gottlieb L.S. Tumoricidal effect of laser energy on experimental and human malignant tumors. N. Engl. J. Med. 1965;273:490–492. [PubMed] 2. Maiman T.H. Stimulated optical radiation in ruby. Nature. 1960;187:493–494. 3. Mester E., Ludány G., Sellyei M., Szende B., Total G.J. The simulating effect of low power laser rays on biological systems. Laser Rev. 1968;1:3. 4. Mester E., Szende B., Gartner P. The effect of laser beams on the growth of hair in mice. Radiobiol. Radiother. 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Epub 2016 May 25. # Cognitiveenhancement by transcranial laser stimulation and acute aerobic exercise. Hwang J1, Castelli DM1, Gonzalez-Lima F2. ### Author information 1 Department of Kinesiology and Health Education, University of Texas at Austin, Austin, TX, 78712, USA. 2 Department of Psychology and Institute for Neuroscience, University of Texas at Austin, 108 E. Dean Keeton Stop A8000, Austin, TX, 78712, USA. gonzalezlima@utexas.edu. ### Abstract This is the first randomized, controlled study comparing the cognitive effects of transcranial laser stimulation and acute aerobic exercise on the same cognitive tasks. We examined whether transcranial infrared laser stimulation of the prefrontal cortex, acute high-intensity aerobic exercise, or the combination may enhance performance in sustained attention and working memory tasks. Sixty healthy young adults were randomly assigned to one of the following four treatments: (1) lowlevel laser therapy (LLLT) with infrared laser to two forehead sites while seated (total 8 min, 1064 nm continuous wave, 250 mW/cm(2), 60 J/cm(2) per site of 13.6 cm(2)); (2) acute exercise (EX) of high-intensity (total 20 min, with 10-min treadmill running at 85-90 % VO2max); (3) combined treatment (LLLT + EX); or (4) sham control (CON). Participants were tested for prefrontal measures of sustained attention with the psychomotor vigilance task (PVT) and working memory with the delayed match-to-sample task (DMS) before and after the treatments. As compared to CON, both LLLT and EX reduced reaction time in the PVT [F(1.56)?=?4.134, p?=?0.01, ? (2) ?=?0.181] and increased the number of correct responses in the DMS [F(1.56)?=?4.690, p?=?0.005, ? (2) ?=?0.201], demonstrating a significant enhancing effect of LLLT and EX on cognitive performance. LLLT + EX effects were similar but showed no significantly greater improvement on PVT and DMS than LLLT or EX alone. The transcranial infrared laser stimulation and acute aerobic exercise treatments were similarly effective for cognitiveenhancement, suggesting that they augment prefrontal cognitive functions similarly. . Curr Alzheimer Res. 2015;12(9):860-9. # Cognitive Improvement by Photic Stimulation in a Mouse Model of Alzheimer’s Disease. Zhang Y, Wang F, Luo X, Wang L, Sun P, Wang M, Jiang Y, Zou J, Uchiumi O, Yamamoto R, Sugai T, Yamamoto K, Kato N1. ### Author information • 1Department of Physiology, Kanazawa Medical University, Ishikawa 920-0293, Japan. kato@kanazawa-med.ac.jp. ### Abstract We previously reported that activity of the large conductance calcium-activated potassium (big-K, BK) channel is suppressed by intracellular A? in cortical pyramidal cells, and that this suppression was reversed by expression of the scaffold protein Homer1a in 3xTg Alzheimer’s disease model mice. Homer1a is known to be expressed by physiological photic stimulation (PS) as well. The possibility thus arises that PS also reverses A?-induced suppression of BK channels, and thereby improves cognition in 3xTg mice. This possibility was tested here. Chronic application of 6-hour-long PS (frequency, 2 Hz; duty cycle, about 1/10; luminance, 300 lx) daily for 4 weeks improved contextual and tone-dependent fear memory in 3xTg mice and, to a lesser extent, Morris water maze performance as well. Hippocampal long-term potentiation was also enhanced after PS. BK channel activity in cingulate cortex pyramidal cells and lateral amygdalar principal cells, suppressed in 3xTg mice, were facilitated. In parallel, neuronal excitability, elevated in 3xTg mice, was recovered to the control level. Gene expression of BK channel, as well as that of the scaffold protein Homer1a, was found decreased in 3xTg mice and reversed by PS. It is known that Homer1a is an activity-dependently inducible immediate early gene product. Consistently, our previous findings showed that Homer1a induced by electrical stimulation facilitated BK channels. By using Homer1a knockouts, we showed that the present PS-induced BK channel facilitation is mediated by Homer1a expression. We thus propose that PS might be potentially useful as a non-invasive therapeutic measure against Alzheimer’s disease. BMC Neurosci. 2016 May 18;17(1):21. doi: 10.1186/s12868-016-0259-6. # Comparative assessment of phototherapy protocols for reduction of oxidative stress in partially transected spinal cord slices undergoing secondary degeneration. Ashworth BE1,2, Stephens E1,2, Bartlett CA1, Serghiou S3, Giacci MK1, Williams A3, Hart NS1,4, Fitzgerald M5. ### Author information • 1Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, WA, Australia. • 2Department of Biology and Biochemistry, The University of Bath, Bath, UK. • 3Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK. • 4Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia. • 5Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, WA, Australia. lindy.fitzgerald@uwa.edu.au. ### Abstract #### BACKGROUND: Red/near-infrared light therapy (R/NIR-LT) has been developed as a treatment for a range of conditions, including injury to the central nervous system (CNS). However, clinical trials have reported variable or sub-optimal outcomes, possibly because there are few optimized treatment protocols for the different target tissues. Moreover, the low absolute, and wavelength dependent, transmission of light by tissues overlying the target site make accurate dosing problematic. #### RESULTS: In order to optimize light therapy treatment parameters, we adapted a mouse spinal cord organotypic culture model to the rat, and characterized myelination and oxidative stress following a partial transection injury. The ex vivo model allows a more accurate assessment of the relative effect of different illumination wavelengths (adjusted for equal quantal intensity) on the target tissue. Using this model, we assessed oxidative stress following treatment with four different wavelengths of light: 450 nm (blue); 510 nm (green); 660 nm (red) or 860 nm (infrared) at three different intensities: 1.93 × 10(16) (low); 3.85 × 10(16) (intermediate) and 7.70 × 10(16) (high) photons/cm(2)/s. We demonstrate that the most effective of the tested wavelengths to reduce immunoreactivity of the oxidative stress indicator 3-nitrotyrosine (3NT) was 660 nm. 860 nm also provided beneficial effects at all tested intensities, significantly reducing oxidative stress levels relative to control (p ? 0.05). #### CONCLUSIONS: Our results indicate that R/NIR-LT is an effective antioxidant therapy, and indicate that effective wavelengths and ranges of intensities of treatment can be adapted for a variety of CNS injuries and conditions, depending upon the transmission properties of the tissue to be treated. J Exp Neurosci. 2016 Feb 1;10:1-19. doi: 10.4137/JEN.S33444. eCollection 2016. # Neuroprotective Effects Against POCD by Photobiomodulation: Evidence from Assembly/Disassembly of the Cytoskeleton. Liebert AD1, Chow RT2, Bicknell BT3, Varigos E4. Author information 1University of Sydney, Sydney, NSW, Australia. 2Brain and Mind Institute, University of Sydney, Sydney, NSW, Australia. 3Australian Catholic University, Sydney, NSW, Australia. 4Olympic Park Clinic, Melbourne, VIC, Australia. Abstract Postoperative cognitive dysfunction (POCD) is a decline in memory following anaesthesia and surgery in elderly patients. While often reversible, it consumes medical resources, compromises patient well-being, and possibly accelerates progression into Alzheimer’s disease. Anesthetics have been implicated in POCD, as has neuroinflammation, as indicated by cytokine inflammatory markers. Photobiomodulation (PBM) is an effective treatment for a number of conditions, including inflammation. PBM also has a direct effect on microtubule disassembly in neurons with the formation of small, reversible varicosities, which cause neural blockade and alleviation of pain symptoms. This mimics endogenously formed varicosities that are neuroprotective against damage, toxins, and the formation of larger, destructive varicosities and focal swellings. It is proposed that PBM may be effective as a preconditioning treatment against POCD; similar to the PBM treatment, protective and abscopal effects that have been demonstrated in experimental models of macular degeneration, neurological, and cardiac conditions. Front Neurosci. 2016 Jan 11;9:500. doi: 10.3389/fnins.2015.00500. eCollection 2015. # Turning On Lights to Stop Neurodegeneration: The Potential of Near Infrared Light Therapy in Alzheimer’s and Parkinson’s Disease. Johnstone DM1, Moro C2, Stone J1, Benabid AL2, Mitrofanis J2. Author information 1Department of Physiology, University of Sydney Sydney, NSW, Australia. 2University Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus Grenoble, France. Abstract Alzheimer’s and Parkinson’s disease are the two most common neurodegenerative disorders. They develop after a progressive death of many neurons in the brain. Although therapies are available to treat the signs and symptoms of both diseases, the progression of neuronal death remains relentless, and it has proved difficult to slow or stop. Hence, there is a need to develop neuroprotective or disease-modifying treatments that stabilize this degeneration. Red to infrared light therapy (? = 600-1070 nm), and in particular light in the near infrared (NIr) range, is emerging as a safe and effective therapy that is capable of arresting neuronal death. Previous studies have used NIr to treat tissue stressed by hypoxia, toxic insult, genetic mutation and mitochondrial dysfunction with much success. Here we propose NIr therapy as a neuroprotective or disease-modifying treatment for Alzheimer’s and Parkinson’s patients. J Mol Neurosci. 2014 Jul 4. [Epub ahead of print] # Low-Level Laser Therapy Ameliorates Disease Progression in a Mouse Model of Alzheimer’s Disease. Farfara D1, Tuby H, Trudler D, Doron-Mandel E, Maltz L, Vassar RJ, Frenkel D, Oron U. ### Author information • 1Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. ### Abstract Low-level laser therapy (LLLT) has been used to treat inflammation, tissue healing, and repair processes. We recently reported that LLLT to the bone marrow (BM) led to proliferation of mesenchymal stem cells (MSCs) and their homing in the ischemic heart suggesting its role in regenerative medicine. The aim of the present study was to investigate the ability of LLLT to stimulate MSCs of autologous BM in order to affect neurological behavior and ?-amyloid burden in progressive stages of Alzheimer’s disease (AD) mouse model. MSCs from wild-type mice stimulated with LLLT showed to increase their ability to maturate towards a monocyte lineage and to increase phagocytosis activity towards soluble amyloid beta (A?). Furthermore, weekly LLLT to BM of AD mice for 2 months, starting at 4 months of age (progressive stage of AD), improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. Histology revealed a significant reduction in A? brain burden. Our results suggest the use of LLLT as a therapeutic application in progressive stages of AD and imply its role in mediating MSC therapy in brain amyloidogenic diseases. Front Syst Neurosci. 2014; 8: 36. Published online 2014 Mar 14. doi: 10.3389/fnsys.2014.00036 PMCID: PMC3953713 # Augmentation of cognitive brain functions with transcranial lasers F. Gonzalez-Lima* and Douglas W. Barrett Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, TX, USA *Correspondence: ude.saxetu@amilzelaznog This article was submitted to the journal Frontiers in Systems Neuroscience. Edited by: Mikhail Lebedev, Duke University, USA Reviewed by: Julio C. Rojas, University of Texas Southwestern Medical Center, USA; John Mitrofanis, University of Sydney, Australia Author information ? Article notes ? Copyright and License information ? Received 2014 Jan 31; Accepted 2014 Feb 27. Keywords: cognitive enhancement, cytochrome oxidase, low-level light therapy, brain stimulation, photoneuromodulation Discovering that transcranial infrared laser stimulation produces beneficial effects on frontal cortex functions such as sustained attention, working memory, and affective state has been groundbreaking. Transcranial laser stimulation with low-power density (mW/cm2) and high-energy density (J/cm2) monochromatic light in the near-infrared wavelengths modulates brain functions and may produce neurotherapeutic effects in a nondestructive and non-thermal manner (Lampl, 2007; Hashmi et al., 2010). Barrett and Gonzalez-Lima (2013) provided the first controlled study showing that transcranial laser stimulation improves human cognitive and emotional brain functions. But for the field of low-level light/laser therapy (LLLT), development of a model of how luminous energy from red-to-near-infrared wavelengths modulates bioenergetics began with in vitro and in vivo discoveries in the last 40 years. Previous LLLT reviews have provided extensive background about historical developments, principles and applications (Rojas and Gonzalez-Lima, 2011, 2013; Chung et al., 2012). The purpose of this paper is to provide an update on LLLT’s neurochemical mechanisms supporting transcranial laser stimulation for cognitive-enhancing applications. We will explain first LLLT’s action on brain bioenergetics, briefly describe its bioavailability and dose-response, and finish with its beneficial effects on cognitive functions. Although our focus is on prefrontal-related cognitive functions, in principle LLLT should be able to modulate other brain functions. For example, stimulating different brain regions should affect different functions related to sensory and motor systems. ## Brain bioenergetics The way that near-infrared lasers and light-emitting diodes (LEDs) interact with brain function is based on bioenergetics, a mechanism that is fundamentally different than that of other brain stimulation methods such as electric and magnetic stimulation. LLLT has been found to modulate the function of neurons in cell cultures, brain function in animals, and cognitive and emotional functions in healthy persons and clinical conditions. Photoneuromodulation involves the absorption of photons by specific molecules in neurons that activate bioenergetic signaling pathways after exposure to red-to-near-infrared light. The 600–1150 nm wavelengths allow better tissue penetration by photons because light is scattered at lower wavelengths and absorbed by water at higher wavelengths (Hamblin and Demidova, 2006). Over 25 years ago, it was found that molecules that absorb LLLT wavelengths are part of the mitochondrial respiratory enzyme cytochrome oxidase in different oxidation states (Karu et al., 2005). Thus, for red-to-near-infrared light, the primary molecular photoacceptor of photon energy is cytochrome oxidase (also called cytochrome c oxidase or cytochrome a-a3) (Pastore et al., 2000). Therefore, photon energy absorption by cytochrome oxidase is well-established as the primary neurochemical mechanism of action of LLLT in neurons (Wong-Riley et al., 2005). The more the enzymatic activity of cytochrome oxidase increases, the more metabolic energy that is produced via mitochondrial oxidative phosphorylation. LLLT supplies the brain with metabolic energy in a way analogous to the conversion of nutrients into metabolic energy, but with light instead of nutrients providing the source for ATP-based metabolic energy (Mochizuki-Oda et al., 2002). If an effective near-infrared light energy dose is supplied, it stimulates brain ATP production (Lapchak and De Taboada, 2010) and blood flow (Uozumi et al., 2010), thereby fueling ATP-dependent membrane ion pumps, leading to greater membrane stability and resistance to depolarization, which has been shown to transiently reduce neuronal excitability (Konstantinovic et al., 2013). On the other hand, electromagnetic stimulation directly changes the electrical excitability of neurons. A long-lasting effect is achieved by LLLT’s up-regulating the amount of cytochrome oxidase, which enhances neuronal capacity for metabolic energy production that may be used to support cognitive brain functions. In mice and rats, memory has been improved by LLLT (Michalikova et al., 2008; Rojas et al., 2012a) and by methylene blue, a drug that at low doses donates electrons to cytochrome oxidase (Rojas et al., 2012b). Near-infrared light stimulates mitochondrial respiration by donating photons to cytochrome oxidase, because cytochrome oxidase is the main acceptor of photons from red-to-near-infrared light in neurons. By persistently stimulating cytochrome oxidase activity, transcranial LLLT induces post-stimulation up-regulation of the amount of cytochrome oxidase in brain mitochondria (Rojas et al., 2012a). Therefore, LLLT may lead to the conversion of luminous energy into metabolic energy (during light exposure) and to the up-regulation of the mitochondrial enzymatic machinery to produce more energy (after light exposure). ## Bioavailability and hormetic dose-response The most abundant metalloprotein in nerve tissue is cytochrome oxidase, and its absorption wavelengths are well correlated with its enzymatic activity and ATP production (Wong-Riley et al., 2005). High LLLT bioavailability to the brain in vivo has been shown by inducing brain cytochrome oxidase activity transcranially, leading to enhanced extinction memory retention in normal rats (Rojas et al., 2012a) and improved visual discrimination in rats with impaired retinal mitochondrial function (Rojas et al., 2008). Our LLLT studies utilized varied wavelengths (633–1064 nm), daily doses (1–60 J/cm2), fractionation sessions (1–6), and power densities (2–250 mW/cm2) that identified effective LLLT parameters for rats and humans. For example, we tested in rats the effects of different LLLT doses in vivo on brain cytochrome oxidase activity, at either 10.9, 21.6, 32.9 J/cm2, or no LLLT. Treatments were delivered for 20, 40, and 60 min via four 660-nm LED arrays with a power density of 9 mW/cm2. One day after the LLLT session, brains were extracted, frozen, sectioned, and processed for cytochrome oxidase histochemistry. A 10.9 J/cm2 dose increased cytochrome oxidase activity by 13.6%. A 21.6 J/cm2 dose produced a 10.3% increase. A non-significant cytochrome oxidase increase of 3% was found after the highest 32.9 J/cm2 dose. Responses of brain cytochrome oxidase to LLLT in vivo were characterized by hormesis, with a low dose being stimulatory, while higher doses were less effective. The first demonstration that LLLT increased oxygen consumption in the rat prefrontal cortex in vivo was provided by Rojas et al. (2012a). Oxygen concentration in the cortex of rats was measured using fluorescence-quenching during LLLT at 9 mW/cm2 and 660 nm. LLLT induced a dose-dependent increase in oxygen consumption of 5% after 1 J/cm2 and 16% after 5 J/cm2. Since oxygen is used to form water within mitochondria in a reaction catalyzed by cytochrome oxidase, more cytochrome oxidase activity should lead to more oxygen consumption. LLLT may offer some advantages over other types of stimulation, because LLLT non-invasively targets cytochrome oxidase, a key enzyme for energy production, with induced expression linked to energy demand. Hence LLLT is mechanistically specific and non-invasive, while transcranial magnetic stimulation may be non-specific, prolonged forehead electrical stimulation may produce muscle spasms, and deep brain or vagus nerve stimulations are invasive. ## Cognitive and emotional functions LLLT via commercial low-power sources (such as FDA-cleared laser diodes and LEDs) is a highly promising, affordable, non-pharmacological alternative for improving cognitive function. LLLT delivers safe doses of light energy that are sufficiently high to modulate neuronal functions, but low enough to not result in any damage (Wong-Riley et al., 2005). In 2002, the FDA approved LLLT for pain relief in cases of head and neck pain, arthritis and carpal tunnel syndrome (Fulop et al., 2010). LLLT has been used non-invasively in humans after ischemic stroke to improve neurological outcome (Lampl et al., 2007). It also led to improved recovery and reduced fatigue after exercise (Leal Junior et al., 2010). One LLLT stimulation session to the forehead, as reported by Schiffer et al. (2009), produced a significant antidepressant effect in depressed patients. No adverse side effects were found either immediately or at 2 or 4 weeks after LLLT. Thus, these beneficial LLLT treatments have been found to be safe in humans. Even though LLLT has been regarded as safe and received FDA approval for pain treatment, the use of transcranial lasers for cognitive augmentation should be restricted to research until further controlled studies support this application for clinical use. We used transcranial laser stimulation to the forehead in a placebo-controlled, randomized study, to influence cognitive tasks related to the prefrontal cortex, including a psychomotor vigilance task (PVT) and a delayed match-to-sample (DMS) memory task (Barrett and Gonzalez-Lima, 2013). The PVT assesses sustained attention, with participants remaining vigilant during delay intervals, and pushing a button when a visual stimulus appears on a monitor. Our laser stimulation targeted prefrontal areas which are implicated in the sustained attentional processes of the PVT (Drummond et al., 2005). Similarly, the DMS task engages the prefrontal cortex as part of a network of frontal and parietal brain regions (Nieder and Miller, 2004). Healthy volunteers received continuous wave near-infrared light intersecting cytochrome oxidase’s absorption spectrum, delivered to the forehead using a 1064 nm low-power laser diode (also known as “cold laser”), which maximizes tissue penetration due to its long wavelength, and has been used in humans for other indications. The power density (or irradiance), 250 mW/cm2, as well as the cumulative energy density (or fluence), 60 J/cm2, were the same that showed beneficial psychological effects in Schiffer et al. (2009). This laser exposure produces negligible heat and no physical damage at the low power level used. This laser apparatus is used safely in a clinical setting by the supplier of the laser (Cell Gen Therapeutics, HD Laser Center, Dallas, TX). Reaction time in the PVT was improved by the laser treatment, as shown by a significant pre-post reaction time change relative to the placebo group. The DMS memory task also revealed significant enhancements in measures of memory retrieval latency and number of correct trials, when comparing the LLLT-treated with the placebo group (Figure (Figure1).1). Self-reported positive and negative affective (emotional) states were also measured using the PANAS-X questionnaire before and 2 weeks after laser treatment. As compared to the placebo, treated subjects reported significantly improved affective states. We suggest that this kind of transcranial laser stimulation may serve as a non-invasive and efficacious method to augment cognitive brain functions related to attention, memory, and emotional functions. Cognitive performance in the delayed match-to-sample (DMS) memory task was improved after transcranial infrared stimulation to the right forehead. The DMS task involves presentation of a visual stimulus (grid pattern) on a screen. Then the stimulus disappears, LLLT’s bioenergetics mechanisms leading to cognitive augmentation may also be at play in its neuroprotective effects (Gonzalez-Lima et al., 2013). LLLT’s stimulation of mitochondrial respiration should improve cellular function due to increased metabolic energy, as well as cellular survival after injury, due to the antioxidant effects of increases in cytochrome oxidase and superoxide dismutase (Rojas et al., 2008). Laser transmittance of the 1064-nm wavelength at the forehead LLLT site was estimated in a post-mortem human specimen, which showed that approximately 2% of the light passed through the frontal bone. This yielded an absorption coefficient of a = 0.24, similar to the reported a = 0.22 transmittance through cranial bone for this wavelength (Bashkatov and Genina, 2006). Thus, we estimated that about 1.2 J/cm2 of the 60 J/cm2 LLLT dose applied reached the surface of the prefrontal cortex. This value is similar to 1 J/cm2, the peak effective LLLT dose in neuron cultures for increasing cytochrome oxidase activity (Rojas and Gonzalez-Lima, 2011). ## Conclusions Transcranial absorption of photon energy by cytochrome oxidase, the terminal enzyme in mitochondrial respiration, is proposed as the bioenergetic mechanism of action of LLLT in the brain. Transcranial LLLT up-regulates cortical cytochrome oxidase and enhances oxidative phosphorylation. LLLT improves prefrontal cortex-related cognitive functions, such as sustained attention, extinction memory, working memory, and affective state. Transcranial infrared stimulation may be used efficaciously to support neuronal mitochondrial respiration as a new non-invasive, cognition-improving intervention in animals and humans. This fascinating new approach should also be able to influence other brain functions depending on the neuroanatomical site stimulated and the stimulation parameters used. ## References • Barrett D. 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Chem. 280, 4761–4771 10.1074/jbc.M409650200 [PubMed] Alzheimers Res Ther. 2014; 6(1): 2. Published online Jan 3, 2014. doi: 10.1186/alzrt232 # Photobiomodulation with near infrared light mitigates Alzheimer’s disease- related pathology in cerebral cortex – evidence from two transgenic mouse models. Sivaraman Purushothuman,1,2 Daniel M Johnstone,1,2 Charith Nandasena,1,2 John Mitrofanis,1,3 and Jonathan Stone1,2 Alzheimer’s disease (AD) is a chronic, debilitating neurodegenerative disease with limited therapeutic options; at present there are no treatments that prevent the physical deterioration of the brain and the consequent cognitive deficits. Histopathologically, AD is characterised by neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein and amyloid-beta (A?) plaques [1,2]. The extent of these histopathological features is considered to vary with and to determine clinical disease severity [2]. While the initiating pathogenic events underlying AD are still debated, there is strong evidence to suggest that oxidative stress and mitochondrial dysfunction have important roles in the neurodegenerative cascade [35]. Therefore, it has been proposed that targeting mitochondrial dysfunction could prove valuable for AD therapeutics [6]. One safe, simple yet effective approach to the repair of damaged mitochondria is photobiomodulation with near-infrared light (NIr). This treatment, which involves the irradiation of tissue with low intensity light in the red to near-infrared wavelength range (600 to 1000 nm), was originally pioneered for the healing of superficial wounds [7] but has been recently shown to have efficacy in protecting the central nervous system. While the mechanism of action remains to be elucidated, there is evidence that NIr preserves and restores cellular function by reversing dysfunctional mitochondrial cytochrome c oxidase (COX) activity, thereby mitigating the production of reactive oxygen species and restoring ATP production to normal levels [8,9]. To date, NIr treatment has yielded neuroprotective outcomes in animal models of retinal damage [9,10], traumatic brain injury [11,12], Parkinson’s disease [1315] and AD [16,17]. Furthermore, NIr therapy has yielded beneficial outcomes in clinical trials of human patients with mild to moderate stroke [18] and depression [19]. This treatment represents a promising alternative to drug therapy because it is safe, easy to apply and has no known side-effects at levels even higher than optimal doses [20]. The aim of this study was to assess the efficacy of NIr in mitigating the brain pathology and associated cellular damage that characterise AD. We utilised two mouse models, each manifesting distinct AD-related pathologies: the K3 tau transgenic model, which develops NFTs [21,22]; and the APP/PS1 transgenic model, which develops amyloid plaques [23]. Here, we present histochemical evidence that NIr treatment over a period of 1 month reduces the severity of AD-related pathology and oxidative stress and restores mitochondrial function in brain regions susceptible to neurodegeneration in AD, specifically the neocortex and hippocampus. The findings extend our previous NIr work in models of acute neurodegeneration [13,14] to demonstrate that NIr is also effective in protecting the brain against chronic insults due to AD-related genetic aberrations, a pathogenic mechanism that is likely to more closely model the human neurodegenerative condition.: ## Methods ### Mouse models The K3 transgenic mouse model, originally generated as a model of frontotemporal dementia [21,22], harbours a human tau gene with the pathogenic K369I mutation; expression is driven by the neuron-specific mThy1.2 promoter. This model manifests high levels of hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We commenced our experiments on K3 mice and matched C57BL/6 wildtype (WT) controls at 5 months of age, when significant neuropathology is already present. The APPswe/PSEN1dE9 (APP/PS1) transgenic mouse model, obtained from the Jackson Laboratory (Stock number 004462; Bar Harbor, ME, USA), harbours two human transgenes: the amyloid beta precursor protein gene (APP) containing the Swedish mutation; and the presenilin-1 gene (PS1) containing a deletion of exon 9 [23]. The APP/PS1 mice exhibit increased A? and amyloid plaques by 4 months of age [24] and cognitive deficits by 6 months of age [25]. We commenced our experiments on APP/PS1 mice and matched C57BL/6×C3H WT controls at 7 months of age, when numerous amyloid plaques and associated cognitive deficits are present. Genotyping of mice was achieved by extracting DNA from tail tips through a modified version of the Hot Shot preparation method [26] and amplifying the transgene sequence by polymerase chain reaction. As reported previously, K3 mice were identified using the primers 5-GGGTGTCTCCAATGCCTGCTTCTTCAG-3 (forward) and 5-AAGTCACCCAGCAGGGAGGTGCTCAG-3 (reverse) [21,22] and APP/PS1 mice were genotyped using primers 5-AGGACTGACCACTCGACCAG-3 (forward) and 5-CGGGGGTCTAGTTCTGCAT-3 (reverse) [23]. ### Experimental design For each series of experiments on K3 mice (aged 5 months) or APP/PS1 mice (aged 7 months) there were three experimental groups: untreated WT mice, untreated transgenic mice and NIr-treated transgenic mice (n=5 mice per experimental group for the K3 series, 15 mice in total; n=6 mice per experimental group for the APP/PS1 series, 18 mice in total). Our design did not include a WT control group exposed to NIr because NIr has no detectable impact on the survival and function of cells in normal healthy brain [1315]. Given the consistency of the previous results, use of animals for this extra control group did not seem justified [27]. Mice in the NIr-treated groups were exposed to one 90-second cycle of NIr (670 nm) from a light-emitting device (LED) (WARP 10; Quantum Devices, Barneveld, WI, USA) for 5 days per week over 4 consecutive weeks. Light energy emitted from the LED during each 90-second treatment equates to 4 Joule/cm2; a total of 80 Joule/cm2 was delivered to the skull over the 4 weeks. Our measurements of NIr penetration across the fur and skull of a C57BL/6 mouse indicate that ~2.5% of transmitted light reaches the cortex. For each treatment, the mouse was restrained by hand and the LED was held 1 to 2 cm above the head. The LED light generated no heat and reliable delivery of the radiation was achieved [1315]. For the sham-treated WT, K3 and APP/PS1 groups, animals were restrained in the same way and the device was held over the head, but the light was not switched on. This treatment regime is similar to that used in previous studies where beneficial changes to neuropathology and behavioural signs were reported [1315]. Experimental animals were housed two or more to a cage and kept in a 12-hour light (<5 lux)/dark cycle at 22°C; food pellets and water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the University of Sydney. ### Histology and immunohistochemistry At the end of the experimental period, mice were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg) and perfused transcardially with 4% buffered paraformaldehyde. Brains were post fixed for 3 hours, washed with phosphate-buffered saline and cryoprotected in 30% sucrose/phosphate-buffered saline. Tissue was embedded in OCT compound (ProSciTech, Thuringowa, QLD, Australia) and coronal sections of the neocortex and the hippocampus (between bregma ?1.8 and ?2.1) were cut at 20 ?m thickness on a Leica cryostat (Nussloch, Germany). #### Immunohistochemistry For most antibodies, antigen retrieval was achieved using sodium citrate buffer with 0.1% Triton. Sections were blocked in 10% normal goat serum and then incubated overnight at 4°C with a mouse monoclonal antibody – paired helical filaments-tau AT8, 1:500 (Innogenetics, Ghent, Belgium); 4-hydroxynonenal (4-HNE), 1:200 (JaICA, Fukuroi, Shizuoka, Japan); 8-hydroxy-2?-deoxyguanosine (8-OHDG), 1:200 (JaICA); COX, 1:200 (MitoSciences, Eugene, OR, USA) – and/or a rabbit polyclonal antibody (200 kDa neurofilament, 1:500; Sigma, St. Louis, MO, USA). Sections were then incubated for 3 hours at room temperature in Alexa Fluor-488 (green) and/or Alexa Fluor-594 (red) tagged secondary antibodies specific to host species of the primary antibodies (1:1,000; Molecular Probes, Carlsbad, CA, USA). Sections were then counterstained for nuclear DNA with bisbenzimide (Sigma). Two different but complementary antibodies were used to label A? peptide: 6E10, which recognises residues 1 to 16; and 4G8, which recognises residues 17 to 24. We have previously used these two antibodies in combination to validate A? labelling, demonstrating identical labelling patterns in the rat neocortex and hippocampus [28]. For double labelling using 6E10 antibodies (1:500; Covance, Princeton, NJ, USA) and anti-glial fibrillary acidic protein antibodies (1:1,000; DAKO, Glostrup, Denmark), antigen retrieval was achieved by incubation in 90% formic acid for 10 minutes, and primary antibody incubation was carried out overnight at room temperature. For labelling using the 4G8 (1:500; Covance) antibody, slides were treated with 3% H2O2 in 50% methanol, incubated in 90% formic acid and then washed several times in dH2O before the blocking step, as described previously [28]. After blocking, sections were incubated overnight at room temperature with 4G8 antibody. Sections were then incubated in biotinylated goat anti-mouse IgG for 1 hour followed by ExtrAvidin peroxidase for 2.5 hours. The sections were then washed and developed with 3,3?-Diaminobenzidine. Negative control sections were processed in the same fashion as described above except that primary antibodies were omitted. These control sections were immunonegative. Fluorescent images were taken using a Zeiss Apotome 2, Carl Zeiss, Oberkochen, Germany. Brightfield images were taken using a Nikon Eclipse E800, Nikon Instruments, Melville, NY, USA. #### Histology NFTs were assessed using the Bielschowsky silver staining method, as described previously [21,22]. Briefly, sections were placed in prewarmed 10% silver nitrate solution for 15 minutes, washed and then placed in ammonium silver nitrate solution at 40°C for a further 30 minutes. Sections were subsequently developed for 1 minute and then transferred to 1% ammonium hydroxide solution for 1 minute to stop the reaction. Sections were then washed in dH2O, placed in 5% sodium thiosulphate solution for 5 minutes, washed, cleared and mounted in dibutyl phathalate xylene. As described previously [28], A? plaques were studied by staining with Congo red, a histological dye that binds preferentially to compacted amyloid with a ?-sheet secondary structure [29]. Briefly, sections were treated with 2.9 M sodium chloride in 0.01 M NaOH for 20 minutes and were subsequently stained in filtered alkaline 0.2% Congo red solution for 1 hour. ### Morphological analysis #### Staining intensity and area measurements To quantify the average intensity and area of antibody labelling within the neocortex and hippocampal regions, an integrated morphology analysis was undertaken using MetaMorph software. For each section, the level of nonspecific staining (using an adjacent region of unstained midbrain) was adjusted to a set level to ensure a standard background across different groups. Next, outlines of retrosplenial cortex area 29 and hippocampal CA1 region were traced and the average intensity and area of immunostaining were calculated by the program. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Statistical analyses were performed in Prism 5.0 (Graphpad, La Jolla, CA, USA) using one-way analysis of variance with Tukey’s multiple comparison post test. All values are given as mean±standard error of mean. #### Amyloid-beta plaque measurements Digital brightfield images of 4G8 staining in the neocortical and hippocampal regions (between bregma ?1.8 and ?2.1) were taken at 4× magnification and analysed with Metamorph, Molecular Devices LLC, Sunnyvale, CA, USA. The software was programmed to measure the number of plaques and the average size of plaques after thresholding for colour. The percentage of area covered by plaques (plaque burden) was calculated by multiplying the number of plaques by the average size of plaques, divided by the area of interest, as described previously [30]. The average number of Congo red-positive plaques in the APP/PS1 brain regions was estimated using the optical fractionator method (StereoInvestigator; MBF Science, Williston, VT, USA), as outlined previously [14]. Briefly, systematic random sampling of sites was undertaken using an unbiased counting frame (100 ?m×100 ?m). All plaques that came into focus within the frame were counted. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Plaque numbers and size were analysed using a two-tailed unpaired t test (when variances were equal) or Welch’s t test (when variances were unequal). All values are given as mean±standard error of mean. For all analyses, investigators were blinded to the experimental groups. ## Results Evidence of NIr-induced neuroprotection is presented from the neocortex (retrosplenial area) and the hippocampus (CA1 and subiculum), two cortical regions affected in the early stages of human AD [2]. ### Near-infrared light mitigates the tau pathology of K3 cortex Hyperphosphorylation of the neuronal microtubule stabilising protein tau and the resulting NFTs are much studied features of dementia pathology [2,31]. The K3 mouse model manifests hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We observe strong labelling for hyperphosphorylated tau in the neocortex and the hippocampus at 6 months of age; expression appears to plateau after this age, with similar labelling observed in 12-month-old mice (Figure 1A,B,C,D,E,F). Time course of the natural development of cortical pathology in K3 and APP/PS1 mice. (A), (B), (C), (D), (E), (F) Micrographs of hyperphosphorylated tau labelling (red), using the AT8 antibody, in the neocortex (A to C) and hippocampus (D toF) of untreated In the retrosplenial area of the neocortex there was a significant overall difference in AT8 immunolabelling for tau between the experimental groups, both when considering average intensity of labelling (P<0.01 by analysis of variance; Figure 2A) and labelled area (P<0.01; Figure 2B). Tukeypost hoc testing revealed significant differences between the untreated K3 group and the other two groups; labelling was much stronger and more widespread in K3 mice than WT controls (17-fold higher intensity, P<0.01), and this labelling was reduced by over 70% in NIr-treated mice (P<0.05). Interestingly, there was no significant difference between the WT and K3-NIr groups, suggesting that NIr treatment had reduced hyperphosphorylated tau to control levels in K3 mice. A similar trend was observed when considering the NFT pathology (Figure 2C,D,E). In contrast to WT brain, which showed no NFT-like lesions (Figure 2C), the K3 brain contained many ovoid shaped NFT-like lesions (that is, spheroids; Figure 2D). Such structures were less frequent in the K3-NIr brain (Figure 2E). Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the neocortex of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error bars Similar effects were observed in the hippocampus (Figure 3). There was a significant overall difference between the experimental groups in AT8 immunolabelling of the CA1 pyramidal cells (P<0.01). As for the neocortex, K3 mice showed far greater labelling than WT mice (17-fold higher intensity, P<0.01) and this was reduced over 65% by NIr treatment (P<0.01). Again, there were no significant differences between the WT and K3-NIr groups (P>0.05). Bielschowsky silver staining of the subiculum (Figure 3C,D,E) revealed axonal swellings and spheroids in the hippocampal region of K3 mice (Figure 3D), which were less pronounced in mice from the K3-NIr group (Figure 3E). No pathology was observed in the hippocampus of WT mice (Figure 3C). Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the hippocampus of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error One should note that the large white matter pathways associated with the hippocampus were labelled intensely by silver staining in all three groups (Figure 3C,D,E). This labelling has been described previously and is not associated with any neuropathology [32]. ### Near-infrared light reduces oxidative stress in K3 cortex Oxidative stress and damage are common features of neurodegenerative diseases such as AD, and may be a precursor to neuronal death [35]. We assessed two common markers of oxidative stress: 4-HNE, a toxic end-product of lipid peroxidation that may bind to proteins that then trigger mitochondrial dysfunction and cellular apoptosis in AD [33]; and 8-OHDG, a marker for nuclear and mitochondrial DNA oxidation, which is elevated in AD brains [34]. Overall, 4-HNE immunoreactivity in the neocortex was significantly different between the experimental groups (Figure 4), by both average labelling intensity (P<0.01) and labelled area (P<0.001). As with AT8 labelling above, the K3 group showed a much higher average 4-HNE labelling intensity and area than the WT group (fivefold and 20-fold, respectively) and this labelling was significantly reduced (by 50% and 80%, respectively) in the K3-NIr group. Again, these measures showed no significant differences between the WT and K3-NIr groups (P>0.05). Effect of near-infrared light treatment on oxidative stress markers in the neocortex of K3 mice. (A), (B), (F), (G)Quantification of immunolabelling of two oxidative stress markers, 4-hydroxynonenal (4-HNE; A, B) and 8-hydroxy-2?-deoxyguanosine Similar patterns were observed for 8-OHDG immunoreactivity. Overall, there was a significant difference between the groups for 8-OHDG immunolabelling, by both average intensity (P<0.0001) and labelled area (P<0.0001). Again the K3 group showed significantly higher 8-OHDG labelling intensity and area than the WT group (sixfold and 17-fold, respectively), and the 8-OHDG labelling intensity and area were significantly reduced in the K3-NIr group relative to untreated K3 (65% and 85% reduction, respectively). The intensity and area of 8-OHDG labelling did not differ significantly between the WT and the K3-NIr groups (P>0.05), suggesting that NIr treatment reduces markers of oxidative stress to control levels. The representative photomicrographs of 8-OHDG immunoreactivity in the retrosplenial area (Figure 4H,I,J) reflect the quantitative data, with many 8-OHDG+ structures in the K3 group (Figure 4I) but not in the WT and K3-NIr groups (Figure 4H,J). ### Near-infrared light mitigates mitochondrial dysfunction in K3 cortex We assessed expression patterns of the mitochondrial enzyme COX in the neocortex and the hippocampus as a marker of mitochondrial function. Overall, there were statistically significant differences in the patterns of COX immunoreactivity between the different experimental groups, both in the neocortex and the hippocampus (both P<0.0001; Figure 5). Relative to WT mice, the COX labelling intensity and area were reduced in K3 mice in both the neocortex and the hippocampus (>70% and >75% reductions, respectively). The K3-NIr mice showed a significant recovery of COX immunoreactivity relative to untreated K3 mice in both the neocortex (>1.7-fold increase, P<0.05) and the hippocampus (>3.4-fold increase, P<0.001). However, recovery was not complete, with K3-NIr mice having significantly lower COX immunoreactivity than WT mice in the neocortex (~50%, P<0.001) and significantly lower COX labelling intensity (~20%, P<0.05) in the hippocampus. These two groups did not differ significantly in COX labelling area in the hippocampus (P>0.05). Effect of near-infrared light treatment on cytochrome coxidase labelling in the neocortex and hippocampus of K3 mice. (A), (B), (F), (G) Quantification of immunolabelling of the mitochondrial marker cytochrome c oxidase (COX) in the neocortex retrosplenial ### Near-infrared mitigates amyloid pathology in APP/PS1 cortex Along with NFTs, A? plaques are considered a primary pathological hallmark of AD and A? load is often used as a marker of AD severity [1,35]. We assessed the distribution of A? plaques and more immature forms of the A? peptide in the neocortex and hippocampus of APP/PS1 mice aged 7 months; this age is after the first signs of intracellular A? within cells (at 3 months; Figure 1G) and extracellular A? plaques (at 4.5 and 12 months; Figure 1H and ?and1I,1I, respectively). Three quantitative measures of plaque pathology were used: percentage plaque burden, average plaque size and number of plaques. Immunohistochemical labelling with the anti-A? antibody 4G8 revealed a significant reduction in percentage plaque burden (Figure 6A,D), average plaque size (Figure 6B,E) and number of plaques (Figure 6C,F) in both the neocortex and the hippocampus of NIr-treated APP/PS1 mice relative to untreated APP/PS1 controls. Percentage plaque burden was reduced by over 40% in the neocortex (Figure 6A; P<0.001) and over 70% in the hippocampus (Figure 6D; P<0.01), average plaque size was reduced 25% in the neocortex (Figure 6B) and 30% in the hippocampus (Figure 6E), and the number of plaques was reduced by over 20% in the neocortex (Figure 6C) and by over 55% in the hippocampus (Figure 6F; all P<0.05). Effect of near-infrared light on amyloid-beta and plaque pathology in APP/PS1 mice. (A), (B), (C), (D), (E), (F)Quantification of amyloid-beta (A?) 4G8 immunolabelling of amyloid plaques in the neocortex (A, B, C) and hippocampus (D, E, F), based The photomicrographs of the 4G8 immunoreactivity in Figure 6 reflect the quantitative data described earlier. The WT brain is free of plaques (Figure 6H,K); many 4G8+ plaques (arrows) are present in the neocortex (Figure 6I) and the hippocampus (Figure 6L) of untreated APP/PS1 mice, and fewer plaques are present in NIr-treated APP/PS1 mice (Figure 6J,M). Comparable immunolabelling was achieved using the 6E10 anti-A? antibody (data not shown). A similar but less pronounced trend was observed when staining with Congo red (Figure 7), which stains only mature plaques. Mean counts of plaques in the neocortex (Figure 7A) and the hippocampus (Figure 7B) of NIr-treated APP/PS1 brains were lower than mean counts in untreated APP/PS1 brains (reductions >30%). However, the differences did not reach statistical significance; given the findings described above with the 4G8 and 6E10 anti-A? antibodies, this suggests that NIr may have greatest effect on recently formed A? deposits. The micrographs in Figure 7 show that mature plaques were absent from the WT brain (Figure 7C,D) but were present in the neocortex (Figure 7E) and hippocampus (Figure 7F) of untreated APP/PS1 brains. There appeared to be fewer plaques in the NIr-treated APP/PS1 brains (Figure 7G,H). Effect of near-infrared light on Congo red-positive plaque numbers in APP/PS1 mice. (A), (B) Quantification of Congo red-positive plaque counts in the neocortex (A) and hippocampus(B). All error bars indicate standard error of the mean. (C), (D), (E), ## Discussion Using two mouse models with distinct AD-related pathologies (tau pathology in K3, amyloid pathology in APP/PS1), we report evidence that NIr treatment can mitigate the pathology characteristic of AD as well as reduce oxidative stress and restore mitochondrial function in brain regions affected early in the disease. Further, the extent of mitigation – to levels less than at the start of treatment – suggests that NIr can reverse some elements of AD-related pathology. The present results add to our previous findings of NIr-induced neuroprotection in models of toxin-induced acute neurodegeneration (that is, MPTP-induced parkinsonism). When incorporated into the growing body of evidence that NIr can also protect against CNS damage in models of stroke, traumatic brain injury and retinal degeneration [912,36], the findings provide a basis for trialling NIr treatment as a strategy for protection against neurodegeneration from a range of causes. Present evidence is based on the use of multiple methods, immunohistochemical and histological, to demonstrate pathological features (for example, 4G8 antibody labelling and Congo red staining for amyloid plaques, AT8 antibody labelling and Bielschowsky silver staining for NFTs). ### Relationship to previous studies The present study focused on pathological features considered characteristic of AD, as well as on signs of cellular damage (for example, oxidative stress, mitochondrial dysfunction) that have been demonstrated in AD and in animal models [24]. Our observations in the K3 strain add to previous studies by providing the first evidence in this strain of extensive oxidative damage and mitochondrial dysfunction [27]. Our findings are consistent with previous reports of the effects of red to infrared light on AD pathology in animal models. De Taboada and colleagues assessed the capacity of 808 nm laser-sourced infrared radiation, delivered three times per week over 6 months, to reduce pathology in an APP transgenic model of A? amyloidosis [17]. Treatment led to a reduction in plaque number, amyloid load and inflammatory markers, an increase in ATP levels and mitochondrial function, and mitigation of behavioural deficits. De Taboada and colleagues commenced treatment at 3 months of age, before the expected onset of amyloid pathology and cognitive effects. Similarly, Grillo and colleagues reported that 1,072 nm infrared light, applied 4 days per week for 5 months, reduces AD-related pathology in another APP/PS1 transgenic mouse model (TASTPM) [16]. These investigators also initiated light treatment before the onset of pathology, at 2 months of age. Both studies thus provide evidence that infrared radiation can slow the progression of cerebral degeneration in these models. The present results confirm these observations, in two distinct transgenic strains; they also confirm that the wound-healing and neuroprotective effects of red-infrared length do not vary qualitatively with wavelength, over a wide range. ### Evidence of reversal of pathology Previous reports have described the natural history of the K3 [21,22] and APP/PS1 transgenic models [24,37]. Based on these previous reports and our own baseline data (Figure 1), significant brain pathology and functional deficits are present in both models at the ages when we commenced treatment. Our results therefore suggest that significant reversal of pathology has been induced by the NIr treatment. This has implications for clinical practice, where most patients are not diagnosed until pathogenic mechanisms have already been initiated and resultant neurologic symptoms manifest [15,27]. This evidence that AD-related neuropathology can be transient – appear then disappear – is not novel. Garcia-Alloza and colleagues described evidence of the transient deposition of A?, including the formation of plaque-like structures, in a transgenic model of A? deposition [24]. Reversal of such pathology, by interventions such as NIr treatment, may therefore be possible. However our results suggest that reversal may also be limited to recently formed, immature plaques, as we observed a significant NIr-induced reduction in immunolabelling with the 4G8 and 6E10 antibodies but no significant difference in Congo red staining. Because the 4G8 and 6E10 antibodies recognise various forms of A?, while Congo red stains only mature, compacted plaques, a reasonable deduction is that NIr treatment reduces only the transient, recently formed A? deposits, with no substantial effect on mature plaques. As there is still no consensus as to the pathogenic roles of different forms of A?, it is unclear how this might impact on the therapeutic potential of NIr in a clinical setting. ### Mechanisms The mechanisms underlying the neuroprotective actions of red to infrared light are not completely understood. There is considerable evidence that NIr photobiomodulation enhances mitochondrial function and ATP synthesis by activating photoacceptors such as COX and increasing electron transfer in the respiratory chain, while also reducing harmful reactive oxygen species [3840]. NIr photobiomodulation could also upregulate protective factors such as nerve growth factor and vascular endothelial growth factor [41,42] and mesenchymal stem cells [43] that could target specific areas of degeneration. The ability of NIr to reduce the expression of hyperphosphorylated tau, which in turn reduces oxidative stress [44], may be key to its neuroprotective effect. Oxidative stress and free radicals increase the severity of cerebrovascular lesions [45,46], mitochondrial dysfunction [4,47], oligomerisation of A? [5,48] and tauopathies and cell death [48,49] in AD. Considering the brain’s high consumption of oxygen and consequent susceptibility to oxidative stress, mitigating such stressors would probably have a pronounced protective effect [50]. ## Conclusions Overall, our results in two transgenic mouse models with existing AD-related pathology suggest that low-energy NIr treatment can reduce characteristic pathology, oxidative stress and mitochondrial dysfunction in susceptible regions of the brain. These results, when taken together with those in other models of neurodegeneration, strengthen the notion that NIr is a viable neuroprotective treatment for a range of neurodegenerative conditions. We believe this growing body of work provides the impetus to begin trialling NIr treatment as a broad-based therapy for AD and other neurodegenerations. ## Abbreviations A?: Amyloid-beta; AD: Alzheimer’s disease; APP: Amyloid beta precursor protein gene; COX: Cytochrome c oxidase; 4-HNE: 4-hydroxynonenal; LED: Light-emitting diode; NFT: Neurofibrillary tangle; NIr: Near-infrared light; 8-OHDG: 8-hydroxy-2?-deoxyguanosine; PS1: Presenilin 1; WT: Wildtype. ## Competing interests The authors declare that they have no competing interests. ## Authors’ contributions SP undertook the bulk of the experimental work and analysis and wrote the manuscript. DMJ and JM were involved with the analysis of the data and the writing of the manuscript. CN was involved with genotyping and treating the animals. JS was involved in conceiving and designing the study and the writing of the manuscript. All authors read and approved the final manuscript. ## Acknowledgments The authors thank Tenix Corporation, Sir Zelman Cowen Universities Fund and Bluesand Foundation for funding. They are grateful to Prof. Lars Ittner for providing the breeding litter for K369I mice, and to Dr Louise Cole and the Bosch Advanced Microscopy facility for the help with MetaMorph. Sharon Spana was splendid for her technical help. DMJ is supported by a National Health and Medical Research Council of Australia (NHMRC) Early Career Fellowship. ## References • Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;6:353–356. doi: 10.1126/science.1072994. [PubMed] • Braak H, Braak E. Staging of Alzheimer’s disease-related neurofibrillary changes. Neurobiol Aging. 1995;6:271–278. doi: 10.1016/0197-4580(95)00021-6. 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Song S, Zhou F, Chen WR, Xing D. Abstract ABSTRACT: BACKGROUND: Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses. METHODS: To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell[trade mark sign] cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT. RESULTS: Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway. CONCLUSIONS: The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases. Postepy High Med Dosw (Online). 2011 Feb 17;65:73-92. # The role of biological sciences in understanding the genesis and a new therapeutic approach to Alzheimer’s disease. Tegowska E, Wosinska A. Zaklad Toksykologii Zwierz?a, Wydzial Biologii i Nauk o Ziemi, Uniwersytet Mikolaja Kopernika w Toruniu. Abstract The paper contrasts the historical view on causal factors in Alzheimer’s disease (AD) with the modern concept of the symptoms’ origin. Biological sciences dealing with cell structure and physiology enabled comprehension of the role of mitochondrial defects in the processes of formation of neurofibrillary tangles and ?-amyloid, which in turn gives hope for developing a new, more effective therapeutic strategy for AD. It has been established that although mitochondria constantly generate free radicals, from which they are protected by their own defensive systems, in some situations these systems become deregulated, which leads to free radical-based mitochondrial defects. This causes an energetic deficit in neurons and a further increase in the free radical pool. As a result, due to compensation processes, formation of tangles and/or acceleration of ?-amyloid production takes place. The nature of these processes is initially a protective one, due to their anti-oxidative action, but as the amount of the formations increases, their beneficial effect wanes. They become a storage place for substances enhancing free radical processes, which makes them toxic themselves. It is such an approach to the primary causal factor for AD which lies at the roots of the new view on AD therapy, suggesting the use of methylene blue-based drugs, laser or intranasally applied insulin. A necessary condition, however, for these methods’ effectiveness is definitely an earlier diagnosis of the disease. Although there are numerous diagnostic methods for AD, their low specificity and high price, often accompanied by a considerable level of patient discomfort, make them unsuitable for early, prodromal screening. In this matter a promising method may be provided using an olfactory test, which is an inexpensive and non-invasive method and thus suitable for screening, although as a test of low specificity, it should be combined with other methods. Introducing new methods of AD treatment does not mean abandoning the traditional ones, based on enhancing cholinergic transmission. They are valuable as long as the therapy starts when abundant brain inclusions disturb the transmissions.<br /> Photomed Laser Surg. 2010 Oct;28(5):663-7. # Long-term safety of single and multiple infrared transcranial laser treatments in Sprague-Dawley rats. McCarthy TJ, De Taboada L, Hildebrandt PK, Ziemer EL, Richieri SP, Streeter J. Source PhotoThera, Inc., 5925 Priestly Drive, Suite 120, Carlsbad, California, USA. tmccarthy@photothera.com Abstract BACKGROUND AND OBJECTIVE: Growing interest exists in the use of near-infrared laser therapies for the treatment of numerous neurologic conditions, including acute ischemic stroke, traumatic brain injury, Parkinson’s disease, and Alzheimer’s disease. In consideration of these trends, the objective of this study was to evaluate the long-term safety of transcranial laser therapy with continuous-wave (CW) near-infrared laser light (wavelength, 808?±?10?nm, 2-mm diameter) with a nominal radiant power of 70?mW; power density, 2,230?mW/cm(2), and energy density, 268?J/cm(2) at the scalp (10?mW/cm(2) and 1.2?J/cm(2) at the cerebral cortical surface) in healthy Sprague-Dawley rats. MATERIALS AND METHODS: In this study, 120 anesthetized rats received sequential transcranial laser treatments to the right and left parietal areas of the head on the same day (minimum of 5?min between irradiation of each side), on either Day 1 or on each of Days 1, 3, and 5. Sixty anesthetized rats served as sham controls. Rats were evaluated 1 year after treatment for abnormalities in clinical hematology and brain and pituitary gland histopathology. RESULTS: No toxicologically important differences were found in the clinical hematology results between sham-control and laser-treated rats for any hematologic parameters examined. All values fell within historic control reference ranges for aged Sprague-Dawley rats. Similarly, brain and pituitary gland histopathology showed no treatment-related abnormalities or induced neoplasia. CONCLUSIONS: Single and multiple applications of transcranial laser therapy with 808-nm CW laser light at a nominal power density of 10?mW/cm(2) at the surface of the cerebral cortex appears to be safe in Sprague-Dawley rats 1 year after treatment. # The Efficacy of 904 nm Laser Therapy for Alzheimer’s Diseases Kazuyoshi Zenba, Vice president of Kanagawa Acupuncture Massage Association Prof. Masayuki Inoue, Secretary of JLPLTPA Preface Although we had reported about the possible efficacy of low power laser therapy (LPLT) for Senile Dementia(S D) 3 times from 1993 at the annual meetings of Japan Society for Laser Medicine, there was no practically useful treatment found for Alzheimer’s disease(AD) and Parkinson disease and other Senile Dementia even after the start of elderly-care-insurance system in Japan. As we have continued above said laser therapy for SD at home care visit of elderly persons and felt very useful and effective, we would like to report about recent situation of laser therapy for AD patients. Especially recently, the number of Alzheimer’s disease patients is increasing by the arrival of super-aged world in Japan. However the cause of this disease is not known and there is no effective treatment established at present. As to the mechanism of LPLT, its main mechanism is mostly elucidated by the progress in the field of Molecular biology and widely used for the removal of pain, decrease of swelling and treatment of wound. However its application for the treatment of Brain diseases is hardly practiced. We have continued the treatment of Senile Dementia patients by LPL considering it as to be one of practical and effective treatment of this disease LPLT is very useful for the medical treatment of the senile dementia patients at home for the expansion of ADL, pain relief, mitigation of inflammation, prevention of bed sore, the treatment of hemiplegia in a brain blood vessel obstacle and the braking of aggravation of Alzheimer’s disease without any fear of side effects by the irradiation of LPL to the head of patients. It will be not to exaggerate to say LPLT can be one of main treatments of senior patients at home in near future. (Object of study) To study the practical usefulness of LPLT for the treatment of Alzheimer’s disease patients at home in terms of improvement of ADL and QOL and also for the reduction of burden of families of the care of patients. (Method of treatment) 15 Alzheimer’s disease patients, 5 male and 10 female, received irradiation of LPL for 2 minutes at each points, 2-3 times a week for one year. Laser irradiation points were as follows. Acupuncture points established as effective based on long history of Oriental medicine . (1) Acupuncture point to improve blood circulation (2) Acupuncture point for the treatment of stroke (3) Acupuncture point for adjustment of blood pressure (4) Acupuncture point for adjustment of balance of autonomous nerve.( the forehead, the right and left temple, occiput) In addition, the method (based on papers in Russia and Armenia that intravenous LPL irradiation improved the viscosity of blood) of irradiating LPL to the place which touches the pulse of an artery under collarbone was used as an additional medical treatment point. (LPL instrument) [Result] Among evaluation items, cooperativeness and the lack of composure were observed as useful as an effect, the effect appeared half a year after and continued after one year and later on. It was suggested that LPLT was useful for the improvement of orientation disturbance, normalization of clothing and the dress. Because, many families and the care workers talked us LPL was very helpful since the present condition could be maintained, without getting worse. After the start of LPL treatment, It was reported that the coldness of hands and legs of patients vanished and joints and muscular stiffness were also mitigated. Therefore, the joint movable region was also secured comparatively. Also in excretion care, it became very easy to carry out the care of patients. It was able to say about all patients that their expression became quiet and came to show understanding to directions of a care worker. It is suggested by this that LPLT as one of practical treatment of patients at home by the improvement of care power at home. (Discussion) Since the senile-dementia-of-Alzheimer-type has a feature of advance of condition and it was said that condition became gradually critical, we tried this treatment expecting the maintenance of condition, and examination whether there was any delay effect. It is considered to have been suggested at least there was an effect of maintaining present condition in a certain field. About the effect over the brain of laser irradiation, it was reported at the annual meeting of Japan Society for Laser Surgery and Medicine meeting in 1991 by Jun-Ichi Nishimura et al., of Department of Physiology, Yokohama City University School of Medicine. The 780 nm wavelength and 1mW laser irradiation to the inner core of rats made the increase of cerebral blood flows at hippocampus by the amount of about 20% in average (control:15, laser:15). Although after 30 minute it was confirmed having maintained the increase of 10%. In 1992 at the same medical conference, Takayuki Obata et. al., of the same University reported that laser irradiation of 780nm wavelength10mW to the head surface of rats activated cranial nerves activities (control:16, laser:15). These reports suggested the possible usefulness of LPL treatment to Senile Dementia and other brain diseases patients. Unfortunately these findings did not much attention of medical world In Japan. However, recently a possibility that ATP and cellmembrane potential of brain neuron could be controlled specifically by the irradiation of near infrared lasers (830nm wavelength) on the surface of heads of rats was reported by Oda-Mochizuki etc.al.?Ritsumeikan University, Synchrotron Light Life Science Center. It was suggested by this research center that the condition of Epilepsy could be stabilized by Irradiating infrared laser from out side of heads of patients and decreasing the unusual excitement of cerebral neurons and in case of cerebral infarction, the aggravation of progress of Necrosis and Apotosis of cerebral neurons could be stopped by making stabilize the electric potential of cell membrane of cerebral neurons. Development of future research in this field is expected as what supports scientifically the medical treatment of LPL and the result of condition improvements, such as Senile Dementia, brain blood vessel obstacles, hemiplegia and Parkinson patients. Although the wavelength of LPL used for “Examination of the validity of LPL to Senile Dementia Patients” which we announced at the annual meetings of Japan Society for Laser Surgery and Medicine meeting over three years from 1993, was 780nm and out put was10mW, and 1mw. The LPL used for this examination was of the wavelength of 904nm and the peak value of a pulse was 5W and the average output was 5mW. However, the same medical treatment effect was confirmed. Although it is thought that there was no wavelength dependability of laser to the efficacy over the Alzheimer’s diseases of LPL(780,830,904nm lasers are equally effective for pain removal and wound healing), how is it sure enough? A question remains. By this examination, at least following effects were confirmed. Namely (1) the advance of condition of Alzheimer’s diseases has been blocked (2) and the expression of patients changed to smiling from disinterestedness, cooperativeness came out , an understanding came to be shown to a partner (3) We received comments from many families that the care of patients became much easier than before. It is considered that the head irradiation of near infrared laser light makes the cerebral blood flow improve, activates nerve activities and have applied brakes to the advance of the apotosis of brain cells as animal experiments are proving. Since the medical treatment efficacy is seldom acknowledged to middle degree class and a serious patient, although it is hard to call it the fundamental cure for Alzheimer’s disease by the present method, if medical a treatment is started in early stage and continued, it may be possible to call it one of practical cures which can stop subsequent advance of disease. Based on this experience, collecting newest information overseas, research results in the biology field, we will continue to study the possible LPL method for the dramatic cure of Alzheimer’s diseases by changing the wavelength of laser, the output and the irradiation method and also combination with other therapies ## Inspire and deepen your practice! Laser, laser needle acupuncture,light emitting diode and pulsed electromagnetic field therapies are the right tools for healing today’s complex patients and for your practice success. All devices pictured above (and more) will likely be available for you to train and practice with in this course. Learn more about them in the links below. Healing Light Seminars and David Rindge have been practicing, teaching and continually updating our treatment methods and equipment since 2002. Our goal, first and foremost, is to provide you with a foundation for success with energy-based therapies. We will only offer devices we have found to be effective, well made and which we are continuing to use clinically. Yet our goal is to ensure that you learn the parameters and methods for success whether or not you buy from us. . Day 1 focuses on theory, biological effects and essentials for clinical success. You have the opportunity for hands-on practice with state-of-the-art laser, laser needle, led and pemf systems for the treatment of pain, head to toe. In Day 2, you will learn how to apply laser, laser needles, led and pulsed electromagnetic field therapies for aesthetics / dermatology / facial rejuvenation, cardiovascular disease, digestive, ear and eye disorders, gynecology, for hair regrowth, neuropathy, osteoporosis, respiratory disorders, sports medicine and more. ### You will receive Laser Therapy: A Clinical Manualas part of the course. This popular training manual by Blahnik and Rindge presents the theory and clinical application of laser therapy in clearly understandable terms with treatment protocols for more than 40 conditions. Laser Therapy: A Clinical Manual is an important important resource in the course and a79.00 value.  You will also receive treatment protocols for other conditions, updates and much, much more relevant material in this course.

Gain a solid understanding of energy-based therapies.    NCCAOM 322-5, seven hours each day, Saturday and Sunday.    Learn More.