Theralase white paper 2011

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INTRODUCTION TO THE LOW LEVEL LIGHT THERAPY AND THE BIOMECHANISMS OF COLD LASERS (White Paper) 2011 Arkady Mandel, MD, Ph.D., D.Sc. Roger Dumoulin-White, P. Eng. Lothar Lilge, Ph.D.

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Transcript of Theralase white paper 2011

  • 1. INTRODUCTION TO THELOW LEVEL LIGHT THERAPY ANDTHE BIOMECHANISMS OF COLD LASERS(White Paper) Arkady Mandel, MD, Ph.D., D.Sc.Roger Dumoulin-White, P. Eng. Lothar Lilge, Ph.D.2011

2. GlossarySYMBOL TERM DEFINITIONMitochondrial Difference in voltage between the interior and exterior of themtmembrane potentialmitochondrial membrane. Total irradiationttot Total period of light exposure timeEffective attenuation Characterizes how easily a material or medium can be eff penetrated by a beam of light. coefficientReduced scattering Coefficient incorporating both the scattering coefficient s and sthe anisotropy coefficient g. coefficientTransfer of energy from an electromagnetic field to a material Absorption Absorptionor to a molecular entity. AdenosineNucleotide derived from ATP with the liberation of energy thatADP is then used in the performance of chemical work.diphosphateCollection of statistical models, and their associatedprocedures, in which the observed variance in a particularANOVA Analysis-of-variancevariable is partitioned into components attributable todifferent sources of variation.Nucleotide found in the mitochondria of all plant and animal Adenosinecells. It is the major source of energy for cellular reactions, thisATP energy being released during its conversion to ADP. Formula: triphosphateC10H16N5O13P3.Quantified by the difference in integrated area under theBiphasic doseBiphasic dosecurve, whereby efficacy is corelated against dosage at depthCorrelates with the characteristics of the wavelength and theBiphasic doseBiphasic doseamount of energy absorbed by chromophore orresponse response photoacceptor. At tissue depth versus perceived efficacy.Ca2+ ions present at nanomolar levels in the cytosol ofCa2+calcium Calcium mammalian cells, and act in a number of signal transductionpathways as second messengers.An arrangement of atoms capable of absorbing light in theChromophoreChromophoresvisible and invisible spectrum And converting this light energy sto chemical energy.Idiopathic median neuropathy at the carpal tunnel. Patientswith CTS experience numbness, tingling, or burning Carpal tunnelCTS sensations in the thumb and fingers, particularly the index,syndromemiddle fingers, and radial half of the ring fingers which areinnervated by the median nerve.CW Continuous waveElectromagnetic wave of constant amplitude and frequency.Colored, heme-containing proteins that transfer electronsCytochromeCytochromeduring cellular respiration and photosynthesis activated bylight energyProportion of time during which a component, device, or DCDuty cycle system is operated Versus the total time of operation ie. % on% cycleRadiant energy, Q, incident from all upward directions on a Energy density or(E/a) act small sphere divided by the cross-sectional area of that radiant exposure sphere. SI unit is J m2. 1 3. SYMBOLTERM DEFINITIONFood and DrugA US federal agency responsible ensuring safety standards in FDAAdministration the food drug and cosmetics industries At a given point in space, the radiant energy, Q, incident on aFluenceFluence small sphere from all directions divided by the cross-sectional area of that sphere. SI unit is J m2. Total radiant power, P, incident from all directions onto aFluence rate Fluence ratesmall sphere divided by the cross-sectional area of that sphere. SI unit is W m2.Anisotropy ofUsed to characterize luminescence (fluorescence, g phosphorescence) polarization resulting from photoselection. biological tissue Respiratory protein formed during physiological respirationHBDeoxy-hemoglobin when oxygen binds to the heme component of the protein hemoglobin in red blood cells. (i.e. arterial blood) Respiratory protein: hemoglobin without the bound oxygen HBO2Oxy-hemoglobin (see Deoxy-hemoglobin). (i.e. venous blood) HeNe Helium NeonHelium Neon laser source Emitting red laser light at 632.8 nm Light power density Threshold at which a physiological effect begins to be I0produced.threshold Indiom GalliumThis laser has a wavelength in the region of approximatelyInGaAI Aarsenide based 905 nm and pulse duration in the 100 to200 nanoseconds can laser sourcebe achieved. A colorless poisonous gas NO formed by oxidation of nitrogen or ammonia that is present in the atmosphere and also inInducible nitriciNOmammals where it is synthesized from arginine and oxygenoxideand acts as a vasodilator and as a mediator of cell-to-cell communication.Inducible nitric Enzymes that catalyze the production of nitric oxide from L- iNOSarginine.oxide synthase Radiant power, P, of all points under consideration divided by the area of the element. SI unit is W m2. wavelengthsIrradiancePower densityincident from all upward directions on a small element of surfaceStimulation powerIstimPower density used to stimulate therapeutic effects densitiesLight Amplification by Stimulated Source of ultraviolet, visible, or infrared radiation. All lasers Emission of Radiation contain an energized substance that can increase the intensityLASERAcronym of Lightof radiation passing through it. The radiation emitted is Activation by coherent except for superradiance emission. Stimulated Emission Better Definitionof Radiation Light EmittingA semiconductor diode that converts applied voltage to new- LEDs Diodescoherent light Drug-free, non-invasive and safe (FDA recognised) clinicalLow Level Light LLLTapplication of light to a patient to promote tissueTherapyregeneration, reduce inflammation and relieve pain Molar (gram M Concentration of a substance per volume of a solutionmolecule/litre) 2 4. SYMBOLTERM DEFINITION Microwave A device or object that emits coherent microwave radiationAmplification by MASER produced by the natural oscillations of atoms or moleculesStimulated Emissionbetween energy levelsof Radiation Maximum Highest power or energy density (in W/cm2 or J/cm2) of aMPE Permissiblelight source that is considered safe. For eyes and for laser Exposureexposure NicotinamideCompound that aids in the Transfer of electrons to carriers NADHadenine dinucleotide that function in Oxidative phosphorylation. Nuclear factor kappa B (NF[kappa]B) has been implicated in Nuclear factorNf-kBgene regulation related to cell proliferation, apoptosis, kappa B adhesion, immune, and inflammatory cellular responses Drug-free, non-invasive and safe (FDA recognised) clinical application of light to a patient to promote tissue regeneration, reduce inflammation and relieve pain in the NILLLT Near-infrared LLLT region of 700 nm to 950 nm of the electromagnetic spectra. Note: 2500 is far infrared to mid Region of the electromagnetic spectrum extending from about NIRNear Infrared 700 nm to 950 nm. Non-metallic element, it combines with other elements, for O2Oxygen molecule combustion and aerobic respiration. Family of purinergic receptors, G protein-coupled receptors P2 (Y and X) Purinergic receptors stimulated by nucleotides such as ATP, ADP, UTP, UDP and UDP-glucose. Time, during the first transition, that the pulse amplitude reaches a specified fraction (level) of its final amplitude, and PDPulse duration(b) the time the pulse amplitude drops, on the last transition, to the same level Chemical reaction caused by absorption of ultraviolet, visible,Photochemical Photochemicalor infrared radiation (infrared.) Effect produced by photoexcitation resulting partially orPhotothermal Photothermaltotally in the production of heat. Photoexcitation and subsequent events, which lead from onePhotophysicalPhotophysical state to another state of a molecular entity, through radiation and radiationless transitions. No chemical change results. Transmission of light during selected pre-determined short PW Pulsed wavetime intervals.QuantumDiscrete values that a spatially confined system or particle canQuantum energy energytake in a quantum mechanical system. redox redox Relating to reduction oxidation cycle of a mammalian cell Gain (reduction) or loss (oxidation) of electrons by an atom orReductionReduction molecule, as occurs when hydrogen is added to a molecule or oxygen is removed.3 5. SYMBOLTERMDEFINITION Rabbit small clotRSCEM embolic stroke Rabbit in vivo model used to demonstrate NILLLT efficacy. model The dispersion of a beam of particles or of radiation into a Scattering Scattering range of directions as a result of physical interactions. Spatial The existence of a correlation between the phases of waves at Spatial coherence coherence points separated in space at a given time.TENS TranscutaneousUse of electric current produced by a device to stimulate theAdd temporal Electrical Nervenerves for temporary relief of pain by increasing the noise coherence level of nerves Stimulation definition 4 6. ON THE MECHANISMS OF LOW LEVEL LIGHT THERAPY (LLLT)Low level light therapy (LLLT) is a rapidly growing modality used in rehabilitation andphysical therapy. A number of safe and beneficial therapeutic effects of LLLT have beenreported in numerous clinical conditions; however, despite many reports of positivefindings from experiments conducted in-vitro, in animal models and in randomizedcontrolled clinical trials worldwide, the use of this scientifically grounded, non-invasive,anti-inflammatory and regulatory modality has yet to find mainstream adoption by medicaldoctors. The aim of this review is six fold:1) introduce the unfamiliar reader, with some medical background, to the contemporaryconcept of LLLT and its pathophysiological significance,2) review the role of the mitochondrial pathway in the mechanisms of LLLT,3) provide necessary practical guidance based on personal scientific and clinicalexperience,4) assist manufacturers in their research and development,5) help health care practitioners choose and use an adequate light therapy device6) outline the prospects of LLLT as an avenue to treat chronic inflammation and pain and toaid in an effective clinical practice.THE STATE OF THE ARTLow level light therapy (LLLT) is the drug-free, non-invasive and safe (FDA recognised)clinical application of light (usually produced by a low to mid power coherent lasers ornon-coherent light emitting diodes (LEDs) in the range of 1mW 500mW) to a patient topromote tissue regeneration, reduce inflammation and relieve pain. The light is typically ofnarrow spectral range (1 to 40 nm bandwidth) in the visible (red) 600 to 700 nm or nearinfrared (NIR) spectrum (700nm to 950nm), achieving an average power density(irradiance) between .001 to 5 w/cm.Light irradiation is typically applied onto the affected area (the areas where the treatmentis needed) or pain projected area or suggested acupuncture points for a few seconds toseveral minutes, daily, or multiple times per week, for a treatment period of up to 8 to 10weeks depending on chronicity. Thus in treatment frequency and duration LLLT does notdiffer from other modalities, such as thermal heating blankets, electrostimulation orultrasound; however, as we shall see dramatically different in cellular mechanism andefficacy.LLLT is not considered an ablative or destructive procedure on the tissue or cellular leveland employs biomodulation via photophysical and potential photochemical mechanisms,comparable to photosynthesis in plants; whereby, the absorbed light is able to initiate acascade of molecular and functional regulatory changes.The reason why the technique is generally referred to as Low Level Laser Therapy (LLLT)is that the optimal level of energy density delivered to the tissues is low when compared toother forms of laser therapy such as the densities required for ablation, cutting, andthermally coagulating tissue. In general, the power densities used for LLLT are lower than5 7. those needed to produce heating of tissue (i.e. less than 500 mWcm-2, depending onwavelength and tissue type), and the total energy delivered to the treated area iscommonly below 1J cm- . A common reference is the Guidelines for Skin Exposure to LaserLight in the International Standards Manual (IEC-825) which considers exposure below200mW cm-2 as a safe exposure (4) in the visible range, escalating to approximately 500mw/cm in the near infrared range.The use of low levels of visible or near infrared light (LLLT) (5), for reduction ofinflammation, elimination of pain (6), healing of wounds, repair to nerves injuries and (7)prevention of tissue damage by reducing cellular apoptosis (for example, due to ischemiaand reperfusion injury), has been known for fifty years since shortly after the invention oflasers in the early 60s.Despite many reports of positive findings from experiments conducted in-vitro, in-vivo(animal and human models) and in randomized controlled clinical trials, the mechanisms ofaction of LLLT in biological tissue remains not fully elucidated. This likely is due to twomain reasons: firstly the complexity in interpretation of scientific and clinical datagenerated in different labs and clinical settings and secondly variability in the use of lightsources (medical devices) and treatment protocols utilized including, illuminationparameters (such as: wavelength, fluence, power density, pulse structure, etc.) and thetreatment schedule. These factors lead to many machinations of factors that make side toside comparisons difficult.As a result, the original field of Low Level Laser (coherent-monochromatic light) Therapy(LLLT) or Low Level Light Therapy (LLLT) subject has now expanded to includephotobiomodulation and photobiostimulation using non-laser (non-coherent light)instruments.These variation in light sources utilized have led to an increase in the number of negativestudies and created some controversy despite the overwhelming number of positiveclinical results (1). It is noteworthy that many studies have been conducted without properscientific methodology, as all the characteristics of the light emitted by lasers or LEDs mustbe specified if a paper is to be useful. A requirement for a good phototherapy study, usinglow intensity radiation in the visible or near-infrared region, whether from a laser, an LED,or a filtered incandescent lamp is to specify everything about the light source, i.e.,wavelength(s), power, dose, area of exposure, time, etc. There are numerous publishedexperimental and clinical studies that were conducted with good scientific methodology,but unfortunately they did not describe the light source (8); therefore, these studies cannotbe repeated or extended by another author. Such publications are useless for progressingthe field of science of LLLT.In recent years, major advances have been made in understanding the mechanisms thatoperate at the cellular and tissue levels during LLLT. Mitochondria are thought to be themain site for the initial effects of light. The discovery and universal acceptance of thisphenomenon is of particular significance to Laser manufactures, because bothmitochondria membrane lipids and the large mitochondrion transmembrane proteincomplex (cytochrome c oxidase) have absorption spectra and action spectra peaks in thered and near infrared regions of the electromagnetic spectrum matching the emission6 8. spectra of the 660 nm and 905 nm, dual wavelengths of Theralases LLLT devices. Thus aphotochemical activation of pathways involving these transmembrane protein complexesas well as potential subcellular photothermal effects is possible (6).New discoveries in the last decade significantly altered our view on mitochondria. They areno longer viewed as energy-making cellular compartments but rather individual cells-within-the-cell. In particular, it has been suggested that many important cellularmechanisms involving specific enzymes and ion channels, such as nitric oxide synthase(NOS), ATP-dependent K+ (KATP) channels, and poly-(APD-ribose) polymerase (PARP), havea distinct, mitochondrial variant. For example, the intriguing possibility that mitochondriaare significant sources of nitric oxide (NO) via a unique mitochondrial NOS variant hasattracted intense interest among research groups because of the potential for NO to affectfunctioning of the electron transport chain (8)It has been shown that LLLT mediated effect may employ inducible nitric oxide synthase(iNOS) to potentially activate production of nitric oxide (NO) in mitochondria (6). Thediscovery of this phenomenon supports a concept that NO and its derivatives (reactivenitrogen species) have multiple effects on mitochondria that may impact a cells physiologyand the cells cycle (7).It was shown that inducible in mitochondria nitric oxide (iNO) inhibits mitochondrialrespiration via: (A) an acute and reversible inhibition of cytochrome c oxidase by NO incompetition with O2, and (B) irreversible inhibition of multiple sites by reactive nitrogenspecies. iNO stimulates reactive oxygen and nitrogen species production frommitochondria via respiratory inhibition, reaction with ubiquinol and reaction with O2 in themitochondrial membranes.(9) Oxidants/free radicals may also confer physiologicalfunctions and cellular signalling processes (10) due to iNO.According to Brown et al., mitochondria may produce and consume NO and NO stimulatesmitochondrial biogenesis, apparently via upregulation of nucleotides like ATP andtranscriptional factors like nuclear factor kappa B (Nf-kB) (9).Therefore, it can be suggested that the super pulsed 905nm LLLT-induced NO canreprogram cellular function, mainly via oxidative stress and changes of mitochondrialtemperature gradient due to process known as selective photothermolysis andconsequently initiate a cascade of local and systemic therapeutic signalling (6).These signal transduction pathways in turn may lead to increased cell activation and traffic,modulation of regulatory cytokines, growth factors and inflammatory mediators, andexpression of protective (anti-apoptotic) proteins (11; 12).The results of these molecular and cellular changes in animals and patients integrate suchbenefits as increased: healing in chronic wounds, improvements in sports injuries andcarpal tunnel syndrome, pain reduction in arthritis and neuropathies, amelioration ofdamage after heart attacks, stroke, nerve injury and alleviation of chronic inflammationand toxicity (13). 7 9. The LLLT-induced NO may explain 1) generation of reactive oxygen species (ROS),induction of transcription factors and increased ATP production (in cells) 2) modulation ofimmune responses, reduction and prevention of apoptosis, regulation of circulation andangiogenesis (in tissues), and suppression of local and systemic inflammation and pain.LLLT-induced ROS, iNO and ATP mediated signalling cascades are well documented in thepeer-reviewed literature (14;15;16;17;18).More evidence now suggests that LLLT is a rapidly growing modality used in physicaltherapy, chiropractic and sport medicine and increasingly entering mainstream medicine.A full spectrum of potential clinical targets that can be successfully treated by LLLT iscontinuing to grow and at this point is not exhausted. LLLT is used to increase woundhealing and tissue regeneration, to relieve pain and inflammation, to prevent tissue deathand to mitigate degeneration in many neurological indications. We believe that furtheradvances in elucidation of the LLLTs mechanisms will lead to greater acceptance of LLLTas the therapy of choice in the established market segments and additionally as a first lineor adjuvant therapy in other serious medical applications, currently not being consideredtypical indications.A SHORT HISTORY OF LLLTLight therapy is one of the oldest therapeutic methods used by humans; (historically assolar therapy by the Egyptians, Aztecs, Greeks and Romans and later as non-ionising UVBphototherapy of lupus vulgaris for which Niels Ryberg Finsen won the Nobel Prize forPhysiology in 1903 (14; 15).The majority of the authors agreed that the era of LLLT starts in 1967, a few years afterGordon Gould coined the acronym LASER (Light Amplification by Stimulated Emission ofRadiation) and described the essential elements for constructing a laser (1957) and 3 yearsafter the Nobel Prize for Physics (1964) was given to Nikolay Gennadiyevich Basov,Aleksandr Mikhailovich Prokhorov and Charles Hard Townes for the invention of theMASER (microwave amplification by stimulated emission of radiation) and the laser (16).The year 1967 is important in the history of LLLT because of the ground breakingpublication by Endre Mester and his colleagues (from Semmelweis University, Budapest,Hungary). The authors uniquely demonstrated the therapeutic benefit of monochromaticvisible light and commenced the therapeutic laser field of science.The investigators original expectation was to test if laser radiation might cause cancer inmice. Mice were shaved and divided into control and treatment groups. The treatmentgroup received the light treatment with a low powered ruby laser (694 nm) while thecontrol group did not. The treated animals did not develop any malignancies and thetreatment was safe; however, the authors made a surprising observation about more rapiddorsal hair regrowth in the treated group than the untreated group (17). Remarkably, thesame study data can be considered as the first disclosure of potential safety and efficacy ofLLLT, particularly attributed to LLLTs biostimulation abilities. 8 10. Since then, medical treatment with coherent light sources (lasers) or noncoherent light(LEDs) has passed through its experimental stage and is firmly rooted in clinical andscientific documentation. Currently, low-level laser (or light therapy LLLT), also known ascold laser, soft laser, biostimulation or photobiomodulation is recognised by theFDA and practiced as part of physical therapy in many parts of the world (18).LLLT is showing promise in the treatment of a wide variety of medical conditions and hasbeen proven to be clinically safe and beneficial therapeutically in many tissues and organs(Figure 1).Figure 1. The diagram below represents the difference in depth of penetration betweentherapeutic lasers.Currently, the knowledge of LLLT mechanisms continues to expand. Physicians are nowaware of LLLTs potential to induce cellular and tissue effects through; for example,accelerated ATP production and molecular signalling and that LLLT can be very effective inthe treatment of various serious clinical disorders.In clinical use, however, it is often difficult to predict patient response to LLLT. It appearsthat several key features, such as: the LLLT parameters and the treatment regimen(including, irradiance [mW cm-2], radiant exposure [J cm-2], treatment regime (includingthe treatment mode, pulsed vs. CW, and the treatment schedule), light attenuation [cm-2] inthe tissues, etc.), cellular pathophysiological status (including, reduction=oxidation (redoxstate), a disease localisation (depth under the surface [mm]) tissue characteristics(including the tissue scattering parameters), the character and the level of inflammatoryprocess and variability of physiological and clinical conditions in patients, can play acentral role in determining sensitivity (and hence clinical efficacy) to LLLT and may help toexplain intra-individual variability in patient responsiveness to the conducted therapy. 9 11. Various cellular responses to visible and infrared radiation have been studied for decadesin the context of molecular mechanisms of low level non-coherent (non-laser) light andlaser phototherapy (19; 20; 21; 22).It is generally accepted that the mitochondria are the initial site of light action inmammalian cells, and cytochrome c oxidase (the terminal enzyme of the mitochondrialrespiratory chain) is one of the key responsible molecules (23) particular due to its broadabsorption in the visible red and NIR spectrum.THE KEY MITOCHONDRIAL MECHANISMS OF LLLTIt is believed that in mammalian organisms the mitochondrial changes taking place areplaying the essential role in the mechanisms of LLLT and, according to Hamblin andcolleagues (2010), the response of cells to light is determined by the mitochondrialnumber, their activity and membrane potential (24).Mitochondria are the energy-transducing organelles of eukaryotic cells in which fuels thatdrive cellular metabolism (i.e., carbohydrates and fats) are converted into adenosinetriphosphate (ATP) through the electron transport chain and the oxidativephosphorylation system (the respiratory chain, Figure 2(29; 30).Mitochondria are also involved in calcium buffering and the regulation of apoptosis (25).They arose as intracellular symbionts in the evolutionary past, and can be traced to theprokaryote -proteobacterium (26). There are hundreds to thousands of mitochondria percell (27), somewhat dependent on the energy requirement of the individual cell.Structurally, mitochondria have four compartments: the outer membrane, the innermembrane, the intermembrane space, and the matrix (the region inside the innermembrane, see Figure 2, (30; 33; 34).The respiratory chain is located on the inner mitochondrial membrane. It consists of fivemultimeric protein complexes: reduced nicotinamide adenine dinucleotide (NADH)dehydrogenase-ubiquinone oxidoreductase (complex I), succinate dehydrogenase-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c oxidoreductase(complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). Inaddition, the respiratory chain requires 2 small electron carriers, ubiquinone andcytochrome c.Energy generation via ATP synthesis involves two coordinated processes: 1) electrons(hydrogen ions derived from NADH and reduced flavin adenine dinucleotide) aretransported along the complexes to molecular oxygen, resulting in the production of water;and 2) simultaneously, protons (hydrogen ions) are pumped across the mitochondrialinner membrane (from the matrix to the intermembrane space) by complexes I, III, and IV.ATP is generated by the influx of these protons back into the mitochondrial matrix throughcomplex V (ATP synthase (27)) .10 12. Figure 2. Mitochondria and Mitochondrial respiratory chainKaru et al. (2004) proposed that biological effects of visible and near-IR light, inmammalian cells, are initiated via the mitochondrial signalling pathway . The authorsalso suggested that the redox absorbance recorded in the spectral range close to 600900nm changes in living cells (28).These observations are of particular interest because a growing number of recent scientificreports and observations are providing more support to the concept that the functions ofmitochondria go beyond the generation of ATP and the regulation of energy metabolism(29).It has been shown that mitochondria are playing a major role as an integrator of intrinsicand extrinsic cellular signals, which can affect the health and survival of the cell; as well as,may play an essential role in the cell-to-cell communication signalling mechanisms (30).ATP binding to the extracellular surface of P2Y-purinergic receptors, allows release ofcytosolic Ca2+calcium, initiates regulatory gene expression and induces a cascade ofintracellular molecular signalling (31).Both Ca2+ uptake and efflux from mitochondria consume the mitochondrial membranepotential (mt); and, in this way modify the mitochondrial activity (and therefore the ATPsynthesis), which can be regulated by LLLT.From a clinicians point of view (for selection of the most effective parameters andappropriate LLLTs medical devices) this scientific data provides a new practical guidelineand assistance towards a better understanding of the biomolecular mechanisms of LLLT.Recent reports have demonstrated that ATP is a critical signalling molecule that allowscells and tissues throughout the body to communicate with one another (32).11 13. This new aspect of ATP as an intercellular signalling molecule allows broadening of theunderstanding of the cellular and molecular mechanisms of LLLT, and hence providesgrounds for optimisation of LLLT efficacy. It has been shown that neurons and other cellsmay release ATP into muscle, gut, and bladder tissue as a messenger molecule. The specificreceptors (a family of P2, purinergic receptors) for ATP signalling were found andidentified (31; 32).ATP activation of P2 receptors (subtypes P2X and P2Y) can produce various biologicaleffects. A recent publication by Anders et al. (2008), demonstrated that P2Y2 and P2Y11receptors were expressed in near-IR light irradiated normal human neural progenitor cellsin vitro (33). These observations support the notion that the irradiation of cells with near-IRlight in the interval close to 905 nm could be functioning as a surrogate for growth factors.Recent reviews indicate that laboratories worldwide are now racing to turn the data aboutATP as a neurotransmitter into therapies (31).As a neurotransmitter, ATP is directly involved in brain function, sensory reception, andthe neuron system control of muscles and organs. When released by neuronal and non-neuronal cells, it often triggers protective responses, such as bone building and cellproliferation.The LLLT-induced ATPs signalling mechanism also is believed to be involved in its useduring pain therapy (29); it is needless to say that chronic and neuropathic pains are the firstdisorders treated successfully with LLLT many decades ago.The role of ATP as a signalling molecule provides a new basis for explaining the versatilityof LLLT effects (34).Numerous reports indicate that light could regulate gene expression via mitochondrialmechanisms. For example, Hu et al., (2007) have shown that mitochondria play a centralrole in cellular homeostasis, and their homeostatic center is the mitochondrial membranepotential ( mt). The assessment of themt in cells conducted by the authors indicatedthat A2058 cells treated with dosages higher than 0.5 J cm-2 He-Ne laser irradiation at 633nm exhibited a marked increase inmt. They also found upregulation of cytochrome coxidase activity, increased phosphorylation of Jun N-terminal kinase (JNK) and later,activated activator protein-1 (AP-1), which led to increased cell proliferation (35).Mitochondrial membrane potential may play a role in response of cells to light therapy.Huang et al. (2004) used tetramethylrhodamine methyl ester to compare the mitochondriaof six different cell types. They found that the meanmt differed significantly between celltypes, but that the cell area or size also differed, and that a more accurate comparison wasto calculate the integrated mt over the cell cross sectional area. Although fibroblasts hada high measured value of meanmt, when integrated over the whole cell the value wasactually the lowest of the cell types tested because fibroblasts had a much greater surfacearea (36).12 14. Neuronal cells had higher mean mt values in the cell bodies compared to the growthcones. Further work is necessary to determine if meanmt or mean mt per cell areacorrelated with measures of cellular response to light. However, there is more supportiveevidence that cell types with greater overall mitochondrial activity (for instance corticalneurons and cardiomyocytes) do in fact respond well to light. The presented observationsmay suggest that cells with a high level of mitochondrial activity (or highmt) may have ahigher response to light than cells with low mitochondrial activity(37).The majority of authors agree that mitochondria are the key cellular targets, able to initiateLLLT induced biologic events, leading to: upregulation of ATP production, induction ofvarious transcription factors and modulation of pro and anti-inflammatory geneexpression. These effects in turn may lead to cell biostimulation, modulation of the levels ofvarious inflammatory mediators (such as cytokines), release of the growth factors, as wellas, increase in tissue oxygenation and reparation. The results of these biologicalinteractions in animals and humans include such therapeutic effects as: acceleration inhealing of chronic wounds, improvements in sport injuries, reduction of inflammation andpain in arthritis and neuropathies, amelioration of toxicity and tissue damage afterinfectious diseases and heart attacks, and restoration of vascular and neuronal damage (38).THE ROLE OF THE 905 nm NIR COHERENT (LASER) LIGHT IN LLLTAn alternative explanation for LLLT effects (particularly in the near-IR wavelength region)on cells and mitochondria does not rely on specific photon absorption by proteins and/orenzymes and their defined absorption bands leading to photochemistry, but relies more onspecific photon absorption leading to localized photothermal effects, confined to sensitivestructures, such as lipids through non-specific photon absorption. The quantum energy ofphotons at 905 nm is only ~1.3 eV and hence insufficient to achieve an electronic excitationin most biomolecules, including cytochrome c oxidase, making a vibrational or thermalactivation of these target molecules more likely at this wavelength.Since the total energy per photon delivered is small the resulting rise in average tissuetemperature would be insignificant if this energy was evenly distributed over the wholecell. However in the case of coherent laser light the energy is not evenly distributed, butforms a speckle pattern at any given instance in time. When a surface is illuminated by alight wave, according to diffraction theory, each point on an illuminated surface acts as asource of secondary spherical waves. Laser speckle is formed by interference (eitherconstructive or destructive) of waves that have been scattered from each point on theilluminated surface. If the surface is rough enough to create path length differencesexceeding one wavelength, giving rise to phase changes greater than 2 , the amplitude, andhence the intensity, of the resultant light varies randomly, from 0 to twice the averageintensity. As tissue is not static these speckle pattern are varying over time and after lessthan 1/100th of a second the temporal average of the intensity is again established acrossthe tissue.If light of low coherence (i.e. made up of many wavelengths moving out of phase) is used, aspeckle pattern will not normally be observed, because the speckle patterns produced by 13 15. individual wavelengths have different dimensions and will normally cancel one anotherout. The "size" of the speckles is a function of the wavelength of the light, the surface area ofthe laser beam that illuminates the first surface, and the distance between this surface andthe plane where the speckle pattern is formed. In tissue, the diameter of the speckles is ofthe order of the wavelength of light i.e. about 1 micron and thus comparable to the size ofthe mitochondria.While continuous wave light sources speckle pattern are averaged temporally as statedabove they do not do so if the light pulse is shorter than the speckle relaxation time.Thus the hypothesis is that laser speckles produce by super pulsed laser sources canproduce micro-thermal gradients due to inhomogeneous energy absorption that canstimulate or otherwise alter the metabolism within mitochondria (39).This hypothesis would explain reports that some LLLT effects in cells and tissues are morepronounced when coherent pulsed laser light is used than comparable non-coherent lightfrom LED or filtered lamp sources that is of similar wavelength range although notmonochromatic (40). For these micro-thermal gradients to be effective the pulse repetitionrate needs to be slow enough to allow a complete relaxation of these gradients (for micronsized hot spots this relaxation time is in the millisecond range) prior to the generation of anovel speckle pattern generated micro-thermal pattern.Lilge, et al., (2009) used near-IR light at 905 nm (Theralase Inc.) and a high peak power of50W cm-2, suggested a role of localized thermal activation (or selective photothermolysis)within mitochondria in the mechanisms of superpulsed 905nm LLLT (5;48;49).The authors suggest that selective photothermolysis occurs if photon absorption within atarget structure is much higher than in the surrounding tissue, but the pulse duration isshort compared to the thermal relaxation time of the target structure. Two hundred-nanosecond pulses of 905 nm laser light, used by the investigators, exceed the thermalrelaxation time of structures smaller than 200 nm. Hence, according to the authors, the cellmembrane (20 nm) would radiate too much energy to the surrounding area not thethermal energy and thus not achieving elevated temperatures, hence presenting anunlikely target structure.Huttmann and colleagues (1999) suggested that the inverse of the repetition rate of 1/10kHz or 100 microseconds needs to be equivalent to at least several thermal relaxationtimes of the target structure, permitting their cooling between pulses to avoidaccumulation of heat, which would finally exceed the Arrhenius damage integral (41).Therefore, the target structure would be required to be smaller than 3 to 5 m, but largerthan 200 nm.Mitochondria membranes have high amount of lipids with peak absorbance at 900 to930nm, close to the superpulsed 905 nm wavelength used. Their inherent size (ahydrophilic headgroup I o . This increased power can cause a transient tissue heating,particularly at specific biological targets (as discussed above in the mitochondrialmechanisms of LLLT) and in this instance pulsed light could be very important. WhereasCW or PW-generated by not true pulsed lasers (including chopped or gated orpseudo technology), particularly emitted by non-coherent (non laser light sources),causes an undesirable increase in temperature of the intervening and target tissues ororgan.Moreover, a true pulsed light that is used in LLLT has been shown to cause no measurablechange in the temperature of the irradiated area for the same delivered energy density asother non pulsed therapeutic light sources (58).In 2006, Ilic S., et al., found that pulsed light (peak power densities of 750mWcm-2)administered for 120 seconds produced no neurological or tissue damage, whereas anequal power density delivered by CW (for the same number of seconds) caused markedneurological deficits (69).Aside from safety advantages true, pulsed or superpulsed light might simply be moreeffective than CW. The quench period (pulse OFF times) reduces tissue heating,; thereby,allowing the use of potentially much higher peak power densities than those that could besafely used in CW. The higher power densities can change the reaction balances in favour ofa positive signalling effect or through the creation of thermal microgradients changing theanalyte exchange across cellular and subcellular membranes. For example, when CWpower densities at the skin use equal or higher than 2 Wcm-2, doubling the CW powerdensity would only marginally increase the treatment depth while potentially significantlyincreasing the risk of thermal damage; in contrast, peak powers of equal or higher than 5Wcm-2 pulsed using appropriate ON and OFF times might produce little, or no tissueheating. The higher pulsed peak powers can avoid tissue heating problems and improve the30 32. fluence rate at deep tissues, achieving greater treatment depths and hence better, overalltherapeutic efficacy (63; 40).There may be other biological reasons for the improved efficacy of pulsed light (PW) overCW. According to Hashmi J.T., et al., the majority of the pulsed light sources used for LLLThave frequencies in the 2.510,000 Hz range and pulse durations are commonly in therange of a few millisecond. This observation suggests that if there is a biologicalexplanation of the improved effects of pulsed light it is either due to some fundamentalfrequency that exists in biological systems in the range of tens to hundreds of Hz, oralternatively due to some biological process that has a time scale of a few milliseconds (58) .Indeed, the possibility of resonance occurring between the frequency of the light pulsesand the neuronal electromagnetic frequency may in some way explain a number of thebeneficial results with LLLT using true pulsed light (70).In addition, there are several lines of evidence that ion channels are involved in thesubcellular effects of LLLT, playing the critical role in the intercellular signalling induced intissues by LLLT (71).Some channels permit the passage of ions based solely on whether their charge is ofpositive (cationic) or negative (anionic) while others are selective for specific species ofion, such as sodium or potassium. These ions move through the channel pore single filenearly as quickly as the ions move through free fluid. In some ion channels, passagethrough the pore is governed by a gate, which may be opened or closed by chemical orelectrical signals, temperature, or mechanical force, depending on the variety of channel.Ion channels are especially prominent components of the nervous system.Voltage activated ion channels underlie the nerve impulse while transmitted, activated orligand gated channels mediate conduction across the synapses. There is a large body ofliterature on the kinetics of various classes of ion channels but in broad summary it can beclaimed that the time scale or kinetics for opening and closing of ion channels is of theorder of a few milliseconds.For instance, there are several reports indicating that the diverse types of ion channelreceptors have kinetics with timescales that are comparable with the superpulsingtechnology (72; 73), as well potassium and calcium ion channels in the mitochondria and thesarcolemma may be involved in the cellular response to LLLT (74; 75)Several authors have also suggested that there is the possibility that one cellularmechanism of action of LLLT is the photodissociation of nitric oxide from a protein bindingsite (heme or copper center) such as those found in cytochrome c oxidase. If this processoccurs it is likely that the NO would rebind to the same site even in the presence ofcontinuous light. Therefore if the light was pulsed multiple photodissociation events couldoccur, while in CW mode the number of dissociations may be much smaller (58).31 33. THE LIGHT PENETRATION DEPTHThe most important parameter that governs the depth of penetration of laser light intotissue is wavelength. When light at a specific wavelength interacts with particular tissue, itis either absorbed or scattered in various proportions depending on the optical propertiesof the tissue. The tissue characteristics are important for LLLT and all other forms ofphototherapy and medical laser applications, in order to understand the interactionmechanisms between light and tissue. Knowledge concerning light transport in tissue andhence the composition of tissue is essential for proper light dosimetry. In general, both theabsorption (a)and scattering (s)coefficients of living tissues are higher at lowerwavelengths, so near-infrared light (up to about 950 nm) penetrates more deeply thatvisible and UV light. It is often claimed that pulsed lasers penetrate more deeply into tissuethan CW lasers with the same average power density (76).For example, if the depth of tissue at which the intensity of the 905 WM CW laser isreduced to 10% is 1cm, and the power density (irradiance) is 100mW cm-2, the fluence rateremaining at 1 cm below the skin surface is 10mWcm-2 and at 2 cm deep is only 1mWcm-2 .Lets assume that a certain threshold power density (minimum number of photons per unitarea per unit time) at the target tissue that is necessary to induce a biological effect is10mW cm-2. Therefore, the effective penetration depth at CW will be 1 cm.Now, suppose that the laser is pulsed with a 10 millisecond pulse duration at a frequency of1Hz (DC = 1 Hz x 0.010 s = 0.010) and the average power is the same (100mWcm-2 ) .The peak power and peak power densities are now 100 times higher (peak power =average power (DC = average power x 100). Therefore with a peak power density of10Wcm-2 at the skin surface, the tissue depth at which this peak power density isattenuated to the threshold level (I) of 10mWcm-2 is now 3 cm rather than 1 cm in CWmode.But what we have to consider is that the laser is only on for 1% of the time so the totalfluence delivered to the 3-cm depth in pulsed mode is 100 times less than that delivered to1-cm depth in CW mode. However it would be possible to increase the illumination time bya factor of 100 to reach the supposed threshold of fluence as well as the threshold of powerat the 3-cm depth. In reality the increase in effective penetration depth obtained withpulsed or super pulsed lasers is more modest than simple calculations might suggest (dueto non linear distribution of photons).Many applications of LLLT may require deep penetration of the light energy, such as deeptreated conditions like: lower back, neck, and hip joint pain and inflammation. Therefore, ifthe power densities need to be greater than a few mWcm-2 and are required to be deliveredsafely to target tissues > 5 cm below the skin surface, the effective treatment can only beachieved by using superpulsed lasers (58).The data indicates that pulsing and particularly superpulsing play an important role inLLLT especially for clinical applications where deep tissue penetration is required. 32 34. THE USE OF COMBINED LASERS (CW + PW) IN LLLTScientific and clinical data suggest that a relatively high fluence (the light energy densitydelivered to the target tissue) is necessary to attenuate pain, whereas a lower fluencedecreases inflammation. If this is indeed the case, for a number of clinical conditions,including musculoskeletal applications, achieving higher doses at the level of the targettissue may not be ideal.It has also been postulated that successful LLLT treatments of arthritic joints bring benefitnot by reaching the deep target tissue directly but by inhibiting superficial nociceptors. Inother words, the therapy brings relief primarily by attenuating pain perception, as opposedto reduction of deep tissue inflammation (58).It becomes evident that the use of a prescribed dose at the tissue surface is insufficient toreach a desired target dose at depth, as the tissue thickness to the target differs betweenpatients, as do the tissue attenuation coefficients which are based on tissue compositions.Hence, new technology is being developed to, optimise these variables and develop asmart) technology that enables the determination of the power density, Istim and the fluenceat the target tissues depth.These scientific reports, as well as, observations on the complementary biological actionsof the far-red and near-infrared irradiation have led to the beginning of the research anddevelopment of a unique class of platform technologies that combine (CW + PW and SP)LLLT approaches; and after FDA and other health regulatory agencies approvals in Canada,Europe, Latin America and in Asia, to a successful commercialization of the new andeffective phototherapeutic medical devices.The latest medical device technology in the field employs visible red (CW) and nearinfrared super pulse (SP) proprietary technology to comprise a unique and clinicallyvalidated array of multiple probe lasers. As a result of this synchronising therapeuticapproach, benefits offered by this new technology includes: 1) delivery of a well-defined,particular dose of coherent photon energy per treatment given to a patient and 2)implementation of cooperative stimulation of the proximal and distal therapeuticmechanisms, in tissues, to induce bioregulatory responses that mitigate inflammation andpain, as well as, the ability to accelerate the healing in the disease in affected areas andassist in maintaining remission, particularly through delivering energy at Istim > Io to therequired depth.Up until now, in the peer-reviewed literature, there has been remarkably little informationavailable about any laser technology that encompasses these concepts.In Gigo-Benatos study, CW or PW stand alone was compared to combined laser. In short,the combined laser was found to be more effective in stimulating nerve regeneration, thaneither CW or PW alone (77).33 35. The two other studies used a combined laser (CW and PW) to administer laseracupuncture, along with Transcutaneous Electrical Nerve Stimulation (TENS), to patientswith symptoms of pain. Naeser et al., administered this triple therapy to patientssuffering from carpal tunnel syndrome (CTS) (78).Eleven patients with mild-to-moderate symptoms of CTS were selected to participate in thetrial. All participants were previously treated by various methods, but had failed to respondto standard medical or surgical treatment regimens. Subjects were divided into two groups,one of which received sham irradiation and the other received a combined treatment ofLLLT (CW and PW) and TENS. As compared to controls, the treated group experiencedstatistically significant improvement and remained stable for 1to 3 years.The results of this study indicate the therapeutic advances of combined LLLT in thetreatment of CTS.Ceccherelli et al., (1989) administered laser acupuncture to patients suffering frommyofascial pain (79).In this double blinded placebo controlled trial, patients received either the same tripletherapy as in the Naeser et al. study (CW, PW, and TENS) or sham irradiation, every otherday over the course of 24 days. Results were encouraging, with the treatment groupexperiencing a significant improvement in symptoms, both immediately after the treatmentregimen and at a 3-month follow up visit. In both preceding studies, the combined regimenof CW, PW, and TENS was compared to untreated controls, and found to be effective (78).However, neither study compared CW and PW or administered CW, PW, or TENSindividually.Therefore, it is impossible to determine whether stand alone the combination of CW andPW light would have produced similar results, or if the used combined LLLT regimen alongwith TENS was more effective.As described above, the therapeutic optical windows (660 nm and 905 nm) utilised by Thelatest LLLT technologies are correspond to the absorption and the action spectra opticalwindows of the key mitochondria chromophores, such as cytochrome c oxidase and cellularmembrane lipids. Moreover, it is apparent that 660 nm and 905 nm light have an impact onthe mitochondrial chromophores via independent mechanisms and hence the combinationof 660 nm and 905 nm light is considered highly probable to have an additive biologiceffect compared to individual wavelengths. This biologic effect is amplified to achieve theeffective therapeutic outcomes via implementation of the latest LLLT technologycooperative CW and PW stimulation system that target the proximal and distal therapeuticmechanisms, in tissues, to induce bioregulatory responses that effectively modulate localand systemic pathologic manifestations in the LLLT treated patients.The clinical results are in support of the preclinical data indicating the efficacy of this latestfrom of LLLT to mitigate the disease progression and to provide significant relief of thesymptoms and health status improvements in chronically ill patients. 34 36. The recent reported, data of a randomized, blinded and placebo controlled pivotal clinicalstudy,(reference) that resulted in FDA approval of the Theralases LLLT technology,demonstrated that this latest LLLT technology provides significant clinical symptoms reliefand a quality of life improvement in patients with osteoarthritis and untreated injuries tomuscles, ligaments and tendons that were enrolled into the study based on the PhiladelphiaPanel Classification, 2001 (80). All patient data for the VAS parameters were analyzed by theanalysis-of-variance (ANOVA) and, in addition, the VAS parameters were analyzed by theRepeated Measures techniques.The data was statistically analyzed to determine the differences and/or whether asignificant difference existed between the laser and placebo (sham) treatment on ahomogeneous population, and it revealed that the tested LLLT had a statistically significantefficacy compared to placebo and greatly improved the pain level by reducing the VASscores by 52.9% at the 12th visit (p= 0.05) and 54.5% at the 30 day (p= 0.01) follow upevaluation.In conclusion, the overall data confirms the theoretical assumption about the dualmechanisms in bioregulatory actions of this latest LLLT technology, demonstrating that thecomplementary, 660 nm (CW) and 905 nm (PW), synchronising therapeutic effect isachieved via direct photochemical and photophysical cellular events initiated bycooperative stimulation of injured tissues (an abnormal or a pathology projected site) bywell-defined doses of coherent light energy.Considering the multifactorial etiopathogenesis of the majority diseases, it is likely that theefficacy of LLLT is determent by optimal sets of therapeutic wavelengths, which should beoptimised based on the optical properties of treated tissues and regiments of light deliverytargeting particular chromophores at specific tissue depth.35 37. 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