MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke; and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata © MD IN ACUPUNCTURE DOCTORAL Thesis Oiucm medicina alternativa March 2010 References in this Doctoral thesis includes parts of a report for the National Institutes of Health Consensus Development Conference on Acupuncture, November, 1997: Neurological Rehabilitation: Acupuncture and Laser Acupuncture to Treat Paralysis in Stroke and Other Paralytic Conditions (Cerebral Palsy, Spinal Cord Injury, and Peripheral Facial Paralysis - Bell's Palsy) and Pain in Carpal Tunnel Syndrome, by Dr. Margaret A. Naeser, Ph.D., Lic.Ac. (Massachusetts), Dipl.Ac. (NCCAOM) and Michael R. Hamblin Department of Dermatology, Harvard Medical School, BAR 414 Wellman Center for Photomedicine, Massachusetts General Hospital 40 Blossom Street, Boston MA 02114
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MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE Low Level Laser Therapy (LLLT) / Cold Laser for healing & pain relief There is substantial clinical evidence published in peer reviewed medical journals that "cold" Low Level laser (LLLT) Acupuncture therapy can stimulate repair of tissue, reduce inflammation and relieve pain in musculoskeletal disorders. The main areas with good evidence are: Soft Tissue Injuries Joint conditions Back and Neck Pain Spinal Cord Injury Brian Injury & Trauma paralysis in stroke paralysis in head injury multiple sclerosis pseudo bulbar palsy & cerebral palsy in babies children spinal cord injury peripheral facial paralysis (Bell's palsy)coma Other applications such as shingles, post operative pain, also respond well. WHAT IS IT? Laser and LED beams of light can stimulate the cells in the body that repair tissue, reduce inflammation and transmit pain. HOW LLLT / COLD LASER THERAPY WORKS Light can stimulate or inhibit cellular function according to its intensity and the time applied. Lasers and LED's produce intense beams of light at specific wavelengths. When the right wavelength at the right intensity is used at the correct anatomical location for the right amount of time you can stimulate repair, resolve inflammation and reduce pain. One of the primary mechanisms is the effect of light on cytochrome c oxidase and the consequently the unbinding of nitric oxide and release of ATP leading to improved cellular function. For antiinflammatory effects we have found a measurable reduction in PGE2, TNF Alpha and IL-6 in the synovia surrounding damaged tendons and for analgesia high intensity lasers over nerve supply can inhibit fast axonal flow. In addition, high intensity single point lasers can release trigger points and treat acupuncture points instead of needles. This doctoral thesis examines LASER Acupuncture modality.
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The use of low levels of visible or near-infrared (NIR) light for reducing pain, inflammation and edema, promoting healing of wounds, deeper tissues and nerves, and preventing tissue damage has been known for almost forty years since the invention of lasers. Originally thought to be a peculiar property of laser light (soft or cold lasers), the subject has now broadened to include photobiomodulation and photobiostimulation using non-coherent light. Despite many reports of positive findings from experiments conducted in vitro, in animal models and in randomized controlled clinical trials, LLLT remains controversial. This likely is due to two main reasons; firstly,
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the biochemical mechanisms underlying the positive effects are incompletely understood, and secondly, the complexity of rationally choosing amongst a large number of illumination parameters such as wavelength, fluence, power density, pulse structure and treatment timing has led to the publication of a number of negative studies as well as many positive ones. In particular, a biphasic dose response has been frequently observed where low levels of light have a much better effect than higher levels. This introductory review will cover some of the proposed cellular chromophores responsible for the effect of visible light on mammalian cells, including cytochrome c oxidase (with absorption peaks in the NIR), and photoactive porphyrins. Mitochondria are thought to be a likely site for the initial effects of light, leading to increased ATP production, modulation of reactive oxygen species, and induction of transcription factors. These effects in turn lead to increased cell proliferation and migration (particularly by fibroblasts), modulation in levels of cytokines, growth factors and inflammatory mediators, and increased tissue oxygenation. The results of these biochemical and cellular changes in animals and patients include such benefits as increased healing of chronic wounds, improvements in sports injuries and carpal tunnel syndrome, pain reduction in arthritis and neuropathies, and amelioration of damage after heart attacks, stroke, nerve injury, and retinal toxicity. 1. HISTORY In 1967, a few years after the first working laser was invented, Endre Mester in Semmelweis University, Budapest, Hungary wanted to test if laser radiation might cause cancer in mice [1]. He shaved the dorsal hair, divided them into two groups and gave a laser treatment with a low powered ruby laser (694 nm) to one group. They did not get cancer, and to his surprise the hair on the treated group grew back more quickly than the untreated group. This was the first demonstration of “laser biostimulation”. Since then, medical treatment with coherent-light sources (lasers) or noncoherent light (light-emitting diodes, LEDs) has passed through its childhood and adolescence. Currently, low-level laser (or light) therapy (LLLT), also known as “cold laser”, “soft laser”, “biostimulation” or “photobiomodulation” is practiced as part of physical therapy in many parts of the world. In fact, light therapy is one of the oldest therapeutic methods used by humans (historically as solar therapy by Egyptians, later as UV therapy for which Nils Finsen won the Nobel prize in 1904 [2]). The use of lasers and LEDs as light sources was the next step in the technological development of light therapy, which is now applied to many thousands of people worldwide each day. In LLLT, the question is no longer whether light has biological effects, but rather how energy from therapeutic lasers and LEDs work at the cellular and organism levels, and what are the optimal light parameters for different uses of these light sources. One important point that has been demonstrated by multiple studies in cell culture [3], animal models [4] and in clinical studies is the concept of a biphasic dose response when the outcome is compared with the total delivered light energy density (fluence). It has been found that there exists an optimal dose of light for any particular application, and doses lower than this optimum value, or more significantly, larger than the optimum value will have a diminished therapeutic outcome, or for high doses of light a negative outcome may even result. Evidence suggests that both energy density and power density are key biological parameters for the effectiveness of laser therapy, and they may both operate with thresholds (i.e., a lower and an upper threshold for both parameters between which laser therapy is effective, and outside of which laser therapy is too weak to have any effect or so intense that the tissue is inhibited) [5].
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The reason why the technique is termed LOW-level is that the optimum levels of energy density delivered are low when compared to other forms of laser therapy as practiced for ablation, cutting, and thermally coagulating tissue. In general, the power densities used for LLLT are lower than those needed to produce heating of tissue, i.e., less than 100 mW/cm2, depending on wavelength and tissue type. 2. PHYSICAL MECHANISMS According to quantum mechanical theory, light energy is composed of photons or discrete packets of electromagnetic energy. The energy of an individual photon depends only on the wavelength. Therefore, the energy of a “dose” of light depends only on the number of photons and on their wavelength or color (blue photons have more energy than green photons, that have more energy than red, that have more energy than NIR, etc). Photons that are delivered into living tissue can either be absorbed or scattered. Scattered photons will eventually be absorbed or will escape from the tissue in the form of diffuse reflection. The photons that are absorbed interact with an organic molecule or chromophore located within the tissue. Because these photons have wavelengths in the red or NIR regions of the spectrum, the chromophores that absorb these photons tend to have delocalized electrons in molecular orbitals that can be excited from the ground state to the first excited state by the quantum of energy delivered by the photon. According to the first law of thermodynamics, the energy delivered to the tissue must be conserved, and three possible pathways exist to account for what happens to the delivered light energy when low level laser therapy is delivered into tissue. The commonest pathway that occurs when light is absorbed by living tissue is called internal conversion. This happens when the first excited singlet state of the chromophore undergoes a transition from a higher to a lower electronic state. It is sometimes called “radiationless deexcitation”, because no photons are emitted. It differs from intersystem crossing in that, while both are radiationless methods of de-excitation, the molecular spin state for internal conversion remains the same, whereas it changes for intersystem crossing. The energy of the electronically excited state is given off to vibrational modes of the molecule, in other words, the excitation energy is transformed into heat. The second pathway that can occur is fluorescence. Fluorescence is a luminescence or re-emission of light, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. The energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat. The wavelengths involved depend on the absorbance curve and Stokes shift of the particular fluorophore. The third pathway that can occur after the absorption of light by a tissue chromophore, represents a number of processes broadly grouped under an umbrella category of photochemistry. Because of the energy of the photons involved, covalent bonds cannot be broken. However, the energy is sufficient for the first excited singlet state to be formed, and this can undergo intersystem crossing to the longlived triplet state of the chromophore. The long life of this species allows reactions to occur, such as energy transfer to ground state molecular oxygen (a triplet) to form the reactive species, singlet oxygen. Alternatively the chromophore triplet state may undergo electron transfer (probably reduction) to form the radical anion that can then transfer an electron to oxygen to form superoxide. Electron transfer reactions are highly important in the mitochondrial respiratory chain, where the principal chromophores involved in laser therapy are thought to be situated. A third photochemistry
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pathway that can occur after the absorption of a red or NIR photon is the dissociation of a noncovalently bound ligand from a binding site on a metal containing cofactor in an enzyme. The most likely candidate for this pathway is the binding of nitric oxide to the iron-containing and coppercontaining redox centers in unit IV of the mitochondrial respiratory chain, known as cytochrome c oxidase (see below). It should be mentioned that there is another mechanism that has been proposed to account for low level laser effects on tissue. This explanation relies on the phenomenon of laser speckle, which is peculiar to laser light. The speckle effect is a result of the interference of many waves, having different phases, which add together to give a resultant wave whose amplitude, and therefore intensity, varies randomly. Each point on illuminated tissue acts as a source of secondary spherical waves. The light at any point in the scattered light field is made up of waves that have been scattered from each point on the illuminated surface.If the surface is rough enough to create path-length differences exceeding one wavelength, giving rise to phase changes greater than 2 , the amplitude (and hence the intensity) of the resultant light varies randomly. It is proposed that the variation in intensity between speckle spots that are about 1 micron apart can give rise to small but steep temperature gradients within subcellular organelles such as mitochondria without causing photochemistry. These temperature gradients are proposed to cause some unspecified changes in mitochondrial metabolism 3. BIOCHEMICAL MECHANISMS There are perhaps three main areas of medicine and veterinary practice where LLT has a major role to play (Figure 1). These are (i) wound healing, tissue repair and prevention of tissue death; (ii) relief of inflammation in chronic diseases and injuries with its associated pain and edema; (iii) relief of neurogenic pain and some neurological problems. The proposed pathways to explain the mechanisms of LLLT should ideally be applicable to all these conditions.
Figure 1. Schematic representation of the main areas of application of LLLT.
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3.1 Tissue photobiology. The first law of photobiology states that for low power visible light to have any effect on a living biological system, the photons must be absorbed by electronic absorption bands belonging to some molecular chromophore or photoacceptor [6]. One approach to finding the identity of this chromophore is to carry out action spectra. This is a graph representing biological photoresponse as a function of wavelength, wave number, frequency, or photon energy, and should resemble the absorption spectrum of the photoacceptor molecule. The fact that a structured action spectrum can be constructed supports the hypothesis of the existence of cellular photoacceptors and signaling pathways stimulated by light. The second important consideration involves the optical properties of tissue. Both the absorption and scattering of light in tissue are wavelength dependent (both much higher in the blue region of the spectrum than the red), and the principle tissue chromophore (hemoglobin) has high absorption bands at wavelengths shorter than 600 nm. For these reasons, there is a so-called “optical window”. The second important consideration involves the optical properties of tissue. Both the absorption and scattering of light in tissue are wavelength dependent (both much higher in the blue region of the spectrum than the red), and the principle tissue chromophores (hemoglobin and melanin) have high absorption bands at wavelengths shorter than 600 nm. Water begins to absorb significantly at wavelengths greater than 1150 nm. For these reasons, there is a so-called “optical window” in tissue covering the red and NIR wavelengths, where the effective tissue penetration of light is maximized (Figure 2). Therefore, although blue, green and yellow light may have significant effects on cells growing in optically transparent culture medium, the use of LLLT in animals and patients almost exclusively involves red and NIR light (600 - 950 nm).
Figure 2. Optical window in tissue due to reduced absorption of red and NIR wavelengths (6001200 nm) by tissue chromophores. 3.2 Action spectra. It was suggested in 1989 that the mechanism of LLLT at the cellular level was based on the absorption of monochromatic visible and NIR radiation by components of the cellular respiratory chain [7]. The inner mitochondrial membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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cytochrome c reductase (Complex III), cytochrome c oxidase (Complex IV), ATP synthase (Complex V), and two freely diffusible molecules, ubiquinone and cytochrome c, which shuttle electrons from one complex to the next (Figure 3). The respiratory chain accomplishes the stepwise transfer of electrons from NADH and FADH2 (produced in the citric acid or Krebs cycle) to oxygen molecules to form (with the aid of protons) water molecules harnessing the energy released by this transfer to the pumping of protons (H+) from the matrix to the intermembrane space. The gradient of protons formed across the inner membrane by this process of active transport forms a miniature battery. The protons can flow back down this gradient, re-entering the matrix, only through another complex of integral proteins in the inner membrane, the ATP synthase complex.
Figure 3. Structure of the mitochondrial respiratory chain. Absorption spectra obtained for cytochrome c oxidase in different oxidation states were recorded and found to be very similar to the action spectra for biological responses to light. Therefore, it was proposed that cytochrome c oxidase (Cox) is the primary photoacceptor for the red-NIR range in mammalian cells [8] (Figure 4). The single most important molecule in cells and tissue that absorbs light between 630 and 900 nm is Cox (responsible for more than 50% of the absorption greater than 800 nm. Cytochrome C oxidase contains two iron centers, haem a and haem a3 (also referred to as cytochromes a and a3), and two copper centers, CuA and CuB [9] . Fully oxidized cytochrome c oxidase has both iron atoms in the Fe(III) oxidation state and both copper atoms in the Cu(II) oxidation state, while fully reduced cytochrome c oxidase has the iron in Fe(II) and copper in Cu(I) oxidation states. There are many intermediate mixed-valence forms of the enzyme and other coordinate ligands such as CO, CN, and formate can be involved. All the many individual oxidation states of the enzyme have different absorption spectra [10], thus probably accounting for slight differences in action spectra of LLLT that have been reported. A recent paper from Karu’s group [11] gave the following wavelength ranges for four peaks in the LLLT action spectrum: 1) 613.5623.5 nm, 2) 667.5-683.7 nm, 3) 750.7-772.3 nm, 4) 812.5-846.0 nm.
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Figure 4. Structure and mode of action of cytochrome c oxidase.
A study from Pastore et al. [12] examined the effect of He-Ne laser illumination (632.8 nm) on the purified cytochrome c oxidase enzyme, and found increased oxidation of cytochrome c and increased electron transfer. Artyukhov and colleagues found [13] increased enzyme activity of catalase after He-Ne laser illumination. The absorption of photons by molecules leads to electronically excited states, and consequently can lead to an acceleration of electron transfer reactions [14]. More electron transport necessarily leads to the increased production of ATP [15]. The light-induced increase in ATP synthesis and increased proton gradient leads to an increasing activity of the Na+/H+ and Ca2+/Na+ antiporters, and of all the ATP driven carriers for ions, such as Na+/K+ ATPase and Ca2+ pumps. ATP is the substrate for adenyl cyclase, and therefore the ATP level controls the level of cAMP. Both Ca2+ and cAMP are very important second messengers. Ca2+ regulates almost every process in the human body (muscle contraction, blood coagulation, signal transfer in nerves, gene expression, etc.). 3.3 Nitric oxide and LLLT. Light mediated vasodilation was first described in 1968 by Furchgott, in his nitric oxide research that lead to his receipt of a Nobel Prize thirty years later in 1998 [16]. Later studies conducted by other researchers confirmed and extended Furchgott’s early work, and demonstrate the ability of light to influence the localized production or release of NO, and to stimulate vasodilation through the effect NO on cGMP. This finding suggests that properly designed illumination devices may be effective, noninvasive therapeutic agents for patients who would benefit
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from increased localized NO availability. However, the wavelengths that are most effective on this light mediated release of NO are different from those used in LLLT, being in the UV-A (320-400 nm) and blue range [17]. Some wavelengths of light are absorbed by hemoglobin, and that illumination can release the NO from hemoglobin (specifically from the nitrosothiols in the beta chain of the hemoglobin molecule) in red blood cells (RBCs) [18-20] Since RBCs are continuously delivered to the area of treatment, there is a natural supply of NO that can be released from each new RBC that passes under the light source, and is exposed to the appropriate wavelength of photo energy. Since the half life of the NO released under the area of illumination is only 2 to 3 seconds, NO release is very local, preventing the effect of increased NO from being manifested in other portions of the body. Vasodilation from NO is based on its effect on the enzyme guanylate cyclase (GC), which forms cGMP to phosphorylate myosin and relax smooth muscle cells in the vascular system. Once available levels of GC are saturated with NO, or once maximum levels of cGMP are achieved, further vasodilation through illumination will not occur until these biologic compounds return to their pre-illumination status. Again, the wavelengths that have been shown to mediate this effect tend to be in the UV-A and blue ranges, not the red and NIR wavelength ranges that are mainly used for LLLT [21]. The activity of cytochrome c oxidase is inhibited by nitric oxide (NO) [22, 23]. This surprising discovery that the body could poison one of its own enzymes was initially shrugged off as an imperfection [24], but a few years later, several groups reported that mitochondria produced an enzyme that synthesizes NO [25], that was identified as the neuronal isoforms of NO synthase [26]. It was proposed that evolution crafted cytochrome c oxidase to bind not only oxygen, but also NO. The effect of slowing respiration in some locations was to divert oxygen elsewhere in cells and tissues, for instance, NO blocks respiration in the endothelial cells lining blood vessels, and this helps to transfer oxygen into smooth muscle cells in these vessels [27]. This inhibition of mitochondrial respiration by NO can be explained by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of cytochrome c oxidase, and is reversible [28]. It was proposed that laser irradiation could reverse the inhibition of cytochrome c oxidase by NO by photodissociating NO from its binding sites [24, 29]. Because this coordinate binding is much weaker than a covalent bond, this dissociation is possible by visible and NIR light that has insufficient energy to break covalent bonds. The dissociation of NO from Cox will thus increase the respiration rate (“NO hypothesis”) [29]. Light can indeed reverse the inhibition caused by NO binding to cytochrome oxidase, both in isolated mitochondria and in whole cells [30]. Light can also protect cells against NO-induced cell death. These experiments used light in the visible spectrum, with wavelengths from 600 to 630 nm. NIR also seems to have effects on cytochrome oxidase in conditions where NO is unlikely to be present. Tiina Karu provided experimental evidence [29] that NO was involved in the mechanism of the cellular response to LLLT in the red region of the spectrum. A suspension of HeLa cells was irradiated with 600-860 nm, or with a diode laser at 820 nm, and the number of cells attached to a glass matrix was counted after a 30 minute incubation. The NO donors, sodium nitroprusside (SNP), glyceryl trinitrate (GTN), or sodium nitrite (NaNO2), were added to the cellular suspension before or after irradiation. Treating the cellular suspension with SNP before irradiation significantly modifies the action spectrum for the enhancement of the cell attachment property, and eliminates the lightinduced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. Other in vivo studies on the use of 780 nm light for stimulating bone
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healing in rats [31], the use of 804 nm laser to decrease damage inflicted in rat hearts after creation of heart attacks [32], have shown significant increases of NO in illuminated tissues after LLLT. On the other hand, studies have been reported on the use of red and NIR LLLT to treat mice with arthritis caused by intra-articular injection of zymosan [33], and studies with 660 nm laser for strokes created in rats [34]. have both shown a reduction of NO in the tissues. These authors explained this observation by proposing that LLLT inhibited inducible nitric oxide synthase (iNOS). In addition to the cytochrome c oxidase mediated increase in ATP production, other mechanisms may be operating in LLLT. The first of these we will consider is the “singlet-oxygen hypothesis.” Certain molecules with visible absorption bands, like porphyrins lacking transition metal coordination centers [35] and some flavoproteins [36], can be converted into a long-lived triplet state after photon absorption. This triplet state can interact with ground-state oxygen with energy transfer leading to production of a reactive species, singlet oxygen. This is the same molecule utilized in photodynamic therapy (PDT) to kill cancer cells, destroy blood vessels, and kill microbes. Researchers in PDT have known for a long time that very low doses of PDT can cause cell proliferation and tissue stimulation, instead of the killing observed at high doses [37]. The next mechanism proposed was the “redox properties alteration hypothesis” [38]. Alteration of mitochondrial metabolism, and the activation of the respiratory chain by illumination would also increase the production of superoxide anions, O2.-. It has been shown that the total cellular production of O2.- depends primarily on the metabolic state of the mitochondria. Other redox chains in cells can also be activated by LLLT. NADPH-oxidase is an enzyme found on activated neutrophils, and is capable of a non-mitochondrial respiratory burst, and production of high amounts of ROS can be induced [39]. These effects depend on the physiological status of the host organism as well as on radiation parameters. 3.4 Cell signaling. The combination of the products of the reduction potential and reducing capacity of the linked redox couples present in cells and tissues represent the redox environment (redox state) of the cell. Redox couples present in the cell include: nicotinamide adenine dinucleotide (oxidized/ reduced forms) NAD/NADH, nicotinamide adenine dinucleotide phosphate NADP/NADPH, glutathione/glutathione disulfide couple GSH/GSSG, and thioredoxin/ thioredoxin disulfide couple Trx(SH)2/TrxSS [40]. Several important regulation pathways are mediated through the cellular redox state. Changes in redox state induce the activation of numerous intracellular signaling pathways, regulate nucleic acid synthesis, protein synthesis, enzyme activation and cell cycle progression [41]. These cytosolic responses in turn induce transcriptional changes. Several transcription factors are regulated by changes in cellular redox state. Among them redox factor-1 (Ref-1)-dependent activator protein-1 (AP-1) (Fos and Jun), nuclear factor (B (NF-(B), p53, activating transcription factor/cAMP-response element-binding protein (ATF/ CREB), hypoxiainducible factor (HIF)-1 , an HIF-like factor. Figure 5 illustrates the effect of redox-sensitive transcription factors activated after LLLT in causing the transcription of protective gene products. As a rule, the oxidized form of redox-dependent transcription factors have low DNA-binding activity. Ref-1 is an important factor for the specific reduction of these transcription factors. However, it was also shown that low levels of oxidants appear to stimulate proliferation and differentiation of some type of cells [42-44].
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Figure 5. Cell signaling pathways induced by LLLT. It is proposed that LLLT produces a shift in overall cell redox potential in the direction of greater oxidation [45]. Different cells at a range of growth conditions have distinct redox states. Therefore, the effects of LLLT can vary considerably. Cells being initially at a more reduced state (low intracellular pH) have high potential to respond to LLLT, while cells at the optimal redox state respond weakly or do not respond to treatment with light. 4. IN VITRO RESULTS 4.1 Cell types. There is evidence that multiple mammalian and microbial cell types can respond to LLLT. Much of Karu’s work has usedEscherichia coli (a Gram-negative aerobic bacterium) [46] and HeLa cells [47], and a human cervical carcinoma cell line. However, for the clinical applications of LLLT to be validated, it is much more important to study the effects of LLLT on non-malignant cell types likely to be usefully stimulated in order to remedy some disease or injury. For wound healing type studies, these cells are likely to be endothelial cells [48], fibroblasts [49], keratinocytes [50], and possibly some classes of leukocytes. such as macrophages [51] and neutrophils [52]. For pain relief and nerve regrowth studies, these cells will be neurons [53-55] and glial cells [56]. For antiinflammatory and anti-edema applications, the cell types will be macrophages [51], mast-cells [57], neutrophils [58], lymphocytes [59], etc. There is literature evidence forin vitro LLLT effects for most of these cell types. 4.2 Isolated mitochondria. Since the respiratory chain and cytochrome c oxidase are located in mitochondria, several groups have tested the effect of LLLT on preparations of isolated Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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mitochondria. The most popular system to study is the effects of HeNe laser illumination (632.8 nm) of mitochondria isolated from rat liver. Increased proton electrochemical potential and ATP synthesis was found [60]. Increased RNA and protein synthesis was demonstrated after 5 J/cm2 [61]. Pastore et al. [62] found increased activity of cytochrome c oxidase, and an increase in polarographically measured oxygen uptake after 2 J/cm2 of 632.8 nm. A major stimulation in the proton pumping activity, about 55% increase of H+/e- ratio was found in illuminated mitochondria. Yu et al. [14] used 660 nm laser at a power density of 10 mW/cm2 and showed increased oxygen consumption (0.6 J/cm2 and 1.2 J/cm2), increased phosphate potential, and energy charge (1.8 J/cm2and 2.4 J/cm2), and enhanced activities of NADH, ubiquinone oxidoreductase, ubiquinol, ferricytochrome C oxidoreductase, and ferrocytochrome C, and oxygen oxidoreductase (between 0.6 J/cm2, and 4.8 J/cm2). 4.3 LLLT cellular response. The cellular responses observed in vitro after LLLT can be broadly classed under increases in metabolism, migration, proliferation, and increases in synthesis and secretion of various proteins. Many studies report effects on more than one of these parameters. Yu et al. [50] reported on cultured keratinocytes and fibroblasts that were irradiated with 0.5-1.5 J/cm2 HeNe laser (632.8 nm). They found a significant increase in basic fibroblast growth factor (bFGF) release from both keratinocytes and fibroblasts, and a significant increase in nerve growth factor release from keratinocytes. Medium from laser irradiated keratinocytes stimulated [3H]thymidine uptake, and the proliferation of cultured melanocytes. Furthermore, melanocyte migration was enhanced either directly by HeNe laser or indirectly by the medium derived from HeNe laser (632.8 nm) treated keratinocytes. The presence of cellular responses to LLLT at molecular level was also demonstrated [63]. Normal human fibroblasts were exposed for 3 days to 0.88J/cm2 of 628 nm light from a light emitting diode. Gene expression profiles upon irradiation were examined using a cDNA microarray containing 9982 human genes. 111 genes were found to be affected by light. All genes from the antioxidant related category and genes related to energy metabolism and respiratory chain were upregulated. Most of the genes related to cell proliferation were upregulated too. Amongst genes related to apoptosis and stress response, some genes such as JAK binding protein were upregulated, others such as HSP701A, caspase 6 and stress-induced phosphoprotein were downregulated. It was suggested that LLLT stimulates cell growth directly by regulating the expression of specific genes, as well as indirectly by regulating the expression of the genes related to DNA synthesis and repair, and cell metabolism. 5. ANIMAL MODELS There has been a large number of animal models that have been used to demonstrate LLLT effects on a variety of diseases, injuries, and both chronic and acute conditions. In this review, I will only discuss three particular applications for which there are good literature reports of efficacy. 5.1 Wound healing. The literature on LLLT applied to a stimulation of wound healing in a variety of animal models contains both positive and negative studies. The reasons for the conflicting reports, sometimes in very similar wound models, are probably diverse. It is probable that applications of LLLT in animal models will be more effective if carried out on models that have some intrinsic disease state. Although there have been several reports that processes such as wound healing are accelerated by LLLT in normal rodents [3, 34], an alternative approach is to inhibit healing by inducing some specific disease state. This has been done in the case of diabetes, a disease known to
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significantly depress wound healing in patients. LLLT significantly improves wound healing in both diabetic rats [35, 36] and diabetic mice [37, 38]. LLLT was also effective in X-radiation impaired wound healing in mice [39]. A study [64] in hairless mice found improvement in the tensile strength of the HeNe laser (632.8 nm)-irradiated wounds at 1 and 2 weeks. Furthermore, the total collagen content was significantly increased at 2 months, when compared with control wounds. The beneficial effect of LLLT on wound healing can be explained by considering several basic biological mechanisms including the induction of expression cytokines and growth factors known to be responsible for the many phases of wound healing. Firstly, there is a report [65] that HeNe laser (632.8 nm) increased both protein and mRNA levels of IL-1 and IL-8 in keratinocytes. These are cytokines responsible for the initial inflammatory phase of wound healing. Secondly, there are reports [66] that LLLT can upregulate cytokines responsible for fibroblast proliferation and migration, such as bFGF, HGF and SCF. Thirdly, it has been reported [67] that LLLT can increase growth factors such as VEGF, responsible for the neovascularization necessary for wound healing. Fourthly, TGF-ß is a growth factor responsible for inducing collagen synthesis from fibroblasts, and has been reported to be upregulated by LLLT [68]. Fifthly, there are reports [69, 70] that LLLT can induce fibroblasts to undergo transformation into myofibloblasts, a cell type that expresses smooth muscle -actin and desmin, and has the phenotype of contractile cells that hasten wound contraction. 5.2 Neuronal toxicity. Studies from Whelan’s group have explored the use of 670 nm LEDs in combating neuronal damage caused by neurotoxins. Methanol intoxication is caused by its metabolic conversion to formic acid that produces injury to the retina and optic nerve, resulting in blindness. Using a rat model and the electroretinogram as a sensitive indicator of retinal function, they demonstrated that three brief 670 nm LED treatments (4 J/cm2), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. There was a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats, and histopathologic evidence of retinal protection [71]. A subsequent study [72] explored the effects of an irreversible inhibitor of cytochrome c oxidase, potassium cyanide, in primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 µM KCN. It significantly reduced neuronal cell death induced by 300 µM KCN from 83.6 to 43.5%. LED significantly restored neuronal ATP content only at 10 µM KCN, but not at higher concentrations of KCN tested. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (670 nm and 830 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase. 5.3 Nerve regeneration. Animal models have been employed to study LLLT effects in nerve repair [73, 74]. Byrnes et al. [56] used 1,600 J/cm2 of 810-nm diode laser to improve healing and functionality in a T9 dorsal hemisection of the spinal cord in rats. Anders et al. [75] studied LLLT for regenerating crushed rat facial nerves; by comparing 361, 457, 514, 633, 720, and 1064 nm, and found the best response with 162.4 J/cm2 of 633 nm HeNe laser. 6. CLINICAL STUDIES Low-power laser therapy is used by physical therapists to treat a wide variety of acute and chronic musculoskeletal aches and pains, by dentists to treat inflamed oral tissues and to heal diverse ulcerations, by dermatologists to treat edema, non-healing ulcers, burns, and dermatitis, by
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orthopedists to relieve pain and treat chronic inflammations and autoimmune diseases, and by other specialists, as well as general practitioners. Laser therapy is also widely used in veterinary medicine (especially in racehorse-training centers), and in sports-medicine and rehabilitation clinics (to reduce swelling and hematoma, relieve pain, improve mobility, and treat acute soft-tissue injuries). Lasers and LEDs are applied directly to the respective areas (e.g., wounds, sites of injuries) or to various points on the body (acupuncture points, muscle-trigger points). However, one of the most important limitations to advancing the LLLT field into mainstream medical practice is the lack of appropriately controlled and blind clinical trials. The trials should be prospective, placebo controlled, and double blinded, and contain sufficient subjects to allow statistically valid conclusions to be reached. Clinical applications of low-power laser therapy are diverse. The field is characterized by a variety of methodologies, and uses of various light sources (lasers, LEDs) with different parameters (wavelength, output power, continuous-wave or pulsed operation modes, pulse parameters). In recent years, longer wavelengths (~800 to 900 nm) and higher output powers (to 100 mW) have been preferred in therapeutic devices, especially to allow deeper tissue penetration. In 2002, MicroLight Corp received 510K FDA clearance for the ML 830 nm diode laser for the treatment of carpal tunnel syndrome. There were several controlled trials reporting significant improvement in pain, and some improvement in objective outcome measures [76-78]. Since then several light sources have been approved as equivalent to an infrared heating lamp for treating a wide-range of musculoskeletal disorders with no supporting clinical studies. 7. UNRESOLVED QUESTIONS 7.1 Wavelength. This is probably the parameter where there is most agreement in the LLLT community. Wavelengths in the 600-700 nm range are chosen for treating superficial tissue, and wavelengths between 780 and 950 nm are chosen for deeper-seated tissues, due to longer optical penetration distances through tissue. Wavelengths between 700 and 770 nm are not considered to have much activity. Some devices combine a red wavelength with a NIR wavelength on the basis that the combination of two wavelengths can have additive effects, and can also allow the device to be more broadly utilized to treat more diseases. There is of course much more work to be done to define what is the optimum wavelength for the different indications for which LLLT is employed. 7.2 Laser vs non-coherent light. One of the most topical and widely discussed issues in the LLLT clinical community is whether the coherence and monochromatic nature of laser radiation have additional benefits, as compared with more broad-band light from a conventional light source or LED with the same center wavelength and intensity. Two aspects of this problem must be distinguished: the coherence of light itself and the coherence of the interaction of light with matter (biomolecules, tissues). The latter interaction produces the phenomenon known as laser speckle, which has been postulated to play a role in the photobiomodulation interaction with cells and subcellular organelles. It is difficult to design an experiment to directly compare coherent laser light with non-coherent non-laser light for the following reason. Laser light is almost always monochromatic with a bandwidth of 1 nm or less, and it is very difficult to generate light from any other source (even an LED) that has a bandwidth narrower than 10-20 nm, therefore it will be uncertain if observed differences are due to coherent versus non-coherent light, or due to monochromatic versus narrow bandwidth light. 7.3 Dose. Because of the possible existence of a biphasic dose response curve referred to above, choosing the correct dosage of light (in terms of energy density) for any specific medical condition is
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difficult. In addition there has been some confusion in the literature about the delivered fluence when the light spot is small. If 5J of light is given to a spot of 5 mm2, the fluence is 100 J/cm2, which is nominally the same fluence as 100 J/cm2 delivered to 10 cm2, but the total energy delivered in the latter case is 200 times greater. The dose of light that is used depends on the pathology being treated, and in particular upon how deep the light is thought to need to penetrate into the tissue. Doses that are frequently used in the red wavelengths for fairly superficial diseases tend to be in the region of 4 J/cm2 with a range of 1-10 J/cm2. Doses of the NIR wavelengths that tend to be employed for deeper-seated disorders can be higher than these values, i.e., in the 10-50 J/cm2 range. The light treatment is usually repeated either every day or every other day, and a course of treatment can last for periods around two weeks. 7.4 Pulsed or CW. There have been some reports that pulse structure is an important factor in LLLT; for instance Ueda et al. [79, 80] found better effects using 1 or 2 Hz pulses than 8 Hz or CW 830 nm laser on rat bone cells, but the underlying mechanism for this effect is unclear. 7.5 Polarization status. There are some claims that polarized light has better effects in LLLT applications than otherwise identical non-polarized light (or even 90-degree rotated polarized light) [81]. However, it is known that polarized light is rapidly scrambled in highly scattering media such as tissue (probably in the first few hundred µ m), and it therefore seems highly unlikely that polarization could play a role, except for superficial applications to the upper layers of the skin. 7.6 Systemic effects. Although LLLT is mostly applied to localized diseases and its effect is often considered to be restricted to the irradiated area, there are reports of systemic effects of LLLT acting at a site distant from the illumination [82, 83]. It is well known that UV light can have systemic effects [84], and it has been proposed that red and NIR light can also have systemic effects. These have been proposed to be mediated by soluble mediators such as endorphins and serotonin. There is a whole field known as laser acupuncture [85] in which the stimulation of specific acupuncture points by a focused laser beam is proposed to have similar effects at distant locations to the more well known needle acupuncture techniques.
ACTION SPECTRA
THEIR IMPORTANCE for LOW LEVEL LIGHT THERAPY
Tiina Karu Laboratory of Laser Biomedicine Institute of Laser and Information Technologies Russian Academy of Sciences Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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Troitsk 142190, Moscow Region, Russian Federation
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1. INTRODUCTION The radiation wavelengths effective for photobiology range between 300 and 900 nm, i.e., from UV (ultraviolet) to near IR (infrared). Practically all photobiological processes in plants and animals, such as photosynthesis, phototropism, phototaxis, photoperiodism, and vision, utilize this range of radiation. The photoreceptor molecules responsible for these photoresponses have been well studied for decades. The regulation of cellular metabolism by visible light is not a classical topic of photobiology. Only the finding of the existence of action spectra in the region from 330 to 860 nm for the increase of DNA and RNA synthesis rates in mammalian cells, as well as for growth stimulation of eukaryotic and prokaryotic microorganisms recorded in the 1980’s (reviewed in 1), indicated that monochromatic light in the visible-to-near region can be a subtle instrument to regulate cellular metabolism. This finding means that the topic of low level laser phototherapy (or low level light therapy, or laser biostimulation) belongs to photobiology. An action spectrum is a plot of the relative effectiveness of different wavelengths of light in causing a particular biological response, and under ideal conditions, it should mimic the absorption spectrum of the molecule that is absorbing the light, and whose photochemical alteration causes the effect (2). The first action spectra with the aim to prove or disapprove the existence of a so-called laser biostimulation effect at the cellular level were recorded in the early 1980’s (3-5). Recall that laser biostimulation (nowadays called low level laser therapy, low level light therapy, photobiomodulation or laser phototherapy) as a medical treatment goes back into the 1960’s with the use of a He-Ne laser ( = 632.8 nm) for the improvement of the healing of impaired wounds. With the technical progress in the second half of the 20th century, and the advent of the laser, these new light sources found their application in medicine and in therapy. The ruby laser ( = 694 nm), which was the first laser invented in 1960, was used in ophthalmology and dermatology very soon after its appearance. Endre Mester, who is considered as the father of “laser biostimulation”, also used the ruby laser in 1964. But the real boom in the therapeutic use of lasers started soon after the invention of the He-Ne laser in 1961, because the He-Ne laser ( = 632.8 nm) was the first widely available commercial laser. The stimulating effect of light, and red light in particular, was rediscovered when this new light source was used. The observed effects were attributed to the unique, high coherence of the He-Ne laser radiation, although there are no physical grounds for such a conclusion (6). Prof. Mester also used the He-Ne laser in his practice, and performed a lot of pioneering studies at the cellular level. At the same time (end of the 60’s - beginning of the 70’s), the large-scale use of He-Ne lasers in laboratories and clinics started in the USSR (Harkov University, Kazahztan State University in Alma-Ata, Institute of Physics in Minsk, Institute of Oncology Problems in Kiev,
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Institute of Radio Engineering and Electronics in Fryazino, Moscow Region). The spreading of laser biostimulation to various countries started at the end of the 70’s, and got a wider interest (Italy, Japan, United Kingdom, and China) after the appearance of semiconductor medical lasers in the 80’s. The action spectra in the visible-to-near IR region for the biological responses of cultured cells showed that red light at 632.8 nm was not the only wavelength suitable for laser biostimulation (35). These spectra together with the results of experiments using the dichromatic irradiation of cells, and the modification of light effects with chemicals (6-9), showed that “laser biostimulation” is a photobiological phenomenon. These data also allowed the suggestion that the photoacceptor for the stimulation of cell metabolism is the terminal enzyme of the respiratory chain, i.e., cytochrome c oxidase for eukaryotic cells (9), and the cytochrome bd complex for Escherichia coli(10). In the blue spectral region, flavoproteins like NADH-dehydrogenase can work as photoacceptors as well (8). The suggestion that cytochrome c oxidase is the photoacceptor molecule has been recently confirmed in elegant experiments with functionally inactivated primary neurons, proposing that light upregulates this enzyme (11). A surprising circumstance is that the photoacceptors for this phenomenon in eukaryotic cells and in prokaryotic cells (E. coli) appeared to be natural components of the respiratory chain, and not specialized photoreceptor molecules. This is different from the classical photobiological phenomena that utilize specific photoreceptors (chlorophylls, rhodopsins, etc.). On the other hand, it is not surprising from the point of view of the absorbing centers in the photoacceptor (for eukaryotic cells these are Cu and quite probably Fe in cytochrome c oxidase, see Section 3). Transition metals (Cu and Fe) generate electronically excited states under very moderate reaction conditions. Bioorganic photochemistry, a rapidly developing new area of research, is concerned with the biological aspects of transition metal chemistry, and physics under irradiation (12). Last but not least, the activation of some enzymes by light, a closely similar phenomenon, is also known (13, 14). Since life evolved in a world of light, there must be many interactions between biological systems and light, including accommodations for its deleterious effects (15). The initial action spectra will be described in Section 2. The analysis of how the photoacceptor was determined from these spectra, is the topic of Section 3. Section 4 describes the comparison of action and absorption spectra. Section 5 is devoted to a brief analysis of how the signals generated by light quanta in mitochondria are transduced to the cellular organelles, where the initial action spectra were measured (the nucleus). 1. Any graph representing a photoresponse as a function of wavelength , wave number -1, or photon energy e, is called an ACTION SPECTRUM. 2. Action spectroscopy analyzes effects caused by irradiation in order to characterize the pigments involved (called photoacceptors or photoreceptors). 3. After determining the photoacceptor molecule, whos absorption spectrum is mirrored in the initial action spectrum, one can make suggestions about cellular signaling pathways inside a cell between the tentative photoacceptor and the molecule whos activity was measured by action spectroscopy.
2. ACTION SPECTRA for an INCREASE of DNA and RNA SYNTHESIS RATE in CULTURED MAMMALIAN CELLS First at all, let us remember that in eukaryotic cells, DNA and RNA synthesis occur in the nucleus, which does not have chromophores absorbing in the spectral region used for laser phototherapy
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(600-900 nm). One can find the original spectra (including the UV-to blue spectral region) (3-5), and in exact form analyzed by contemporary software, in (16). Below, we will also analyze the action spectrum for the increase in cell attachment to a glass matrix (17). Figure 1 presents five action spectra in the red-to-near IR region for mammalian cells, and Table 1 provides the data of their deconvolution. Original experimental data (4, 5, 17) are presented here together with curve fitting and Lorentzian fitting (16). The mean-square deviation, R2, for every fitting is also shown in Table 1. At the best fitting, R2 =1. Spectra A and B present the stimulation of DNA synthesis rate in log-phase and plateau-phase cultures, respectively. Spectra C and D are the dependencies of stimulation of RNA synthesis rate in log- and plateau-phase cells. These four spectra were recorded using a monochromator MDR-2, with a halogen lamp with the power of 150 W placed in a parabolic reflector (4). The spectral full width at half maximum (FWHM) of the produced light was 14 nm (Figures 1A-D). Light intensity was kept constant (10 W/m2) by varying the voltage across the halogen lamp. The irradiation time was 10 s, and the dose was 100 J/m2. Spectrum E shows an increase in cell attachment to a glass matrix. In this case, the monochromator was a more advanced one, constructed in Institute of Spectroscopy of Russian Academy Science (17). Light parameters for recording of this spectrum were as follows: I = 1.3 W/m2, t = 40 sec, D = 52 J/m2, FWHM 10 nm.
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Figure 1. Action spectra in the region of 580-860 nm for (A) stimulation of DNA synthesis rate in log-phase and (B) plateau-phase cultures; (C) stimulation of RNA synthesis rate in log-phase and (D) plateau-phase cultures; (E) increase of cell attachment to a glass matrix. Experimental curves ( , adapted from 4, 5, 17), curve fittings (solid line), and Lorentzian fittings (dashed line) are shown. Dose 100 J/m2(A-D) or 52 J/m2 (E). Adapted from (16). All five spectra in Figure 1 are characterized by four wide maxima, but the exact peak positions are different (Table 1). The largest differences in peak position can be seen in the near IR region above 800 nm. This peak appears at 812.5 nm in the spectrum A, between 827.5 and 830.7 nm in the spectra C, D, E, and at 846.0 nm, in the spectrum B.
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The maximum in spectra A-E in Figure 1 has peak positions from 750.7 to 772.3 nm (Table 1). In four spectra, this peak is between 761.1 and 772.3 nm, which could be one line in an absorption spectrum. An appearance of this maximum with a peak position at 750.7 nm in spectrum E could probably mirror another line in an absorption spectra. The far-red maximum in the action spectra (Figure 1) has peak positions from 667.5 to 684.5 nm. These peak positions can be divided in to two groups: 667.5, 671.5, and 668.0 nm in one group, and 684.5 and 683.7 nm in the other (Table 1). The red maximum in the action spectra (Figure 1) has peak positions from 613.5 to 623.5 nm (Table 1). It should be noted that in an early action spectrum (3), a peak at 606 nm appeared together with a peak at 632.8 nm (Figure 2A, Table 2). These first two spectra for the stimulation of DNA and RNA synthesis rate were recorded from 570 to 650 nm, using filament lamps with a power of 20 and 90 W, and interference filters (3). With that setup we were not able to keep the intensity of the light equal at all wavelengths. This means that for a constant dose one was forced to use various irradiation times. In our case, the intensity was 1.5 and 0.3 W/m2, and the dose of 80 J/m2 was reached by irradiating the cells from 2 s to 4.5 min.
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Figure 2. Action spectra in the region of 570-650 nm for the stimulation of (A) DNA and (B) RNA synthesis in log-phase cultures at D = 80 J/m2 under conditions where irradiation times were not kept constant. Experimental curves ( , adapted from 3), curve fittings (solid lines), and Lorentzian fittings (dashed lines) are shown. Adapted from (16). This was not a correct measurement of an action spectrum, but this first experiment showed several important features of the biostimulation phenomenon. First, not only He-Ne laser light at 632.8 nm causes “biostimulation”, a similar result was achieved using noncoherent light of the same wavelength. Recall that in year 1982, the medical laser community believed that He-Ne laser radiation had magical beneficial properties. Secondly, after improving the equipment and comparing new action spectra (Figure 1, A-D) to the first spectra presented in Figure 2A, B, we understood that there should be a dependence on light intensity in the far-red region 650-680 nm. Speaking in photobiological terms, the reciprocity rule does not hold. According to the reciprocity (or BunsenRoscoe) rule, a photochemical reaction is directly proportional to the total energy dose, irrespective of the time over which this dose is delivered. However, the reason why in one spectrum (Figure 2A) two red bands appeared, is still obscure. One can only suppose that this is due to irradiation parameters, intensity and irradiation time. Solving this question requires new experiments.
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Finding the absence of reciprocity lead us to perform a special experiment, where the dose was kept constant, but the irradiation time and intensity were varied. These data are presented here to draw attention once more to the threshold-type of behavior of the intensity-dependence that is still not understood, and is often not taken into account in experiments. It appeared that the red maximum in the action spectra is sensitive to light intensity (Figure 3), and that there exists a certain threshold to receive a stimulation effect (in our case, at 4-5 W/m2) (3). The maximum effect occurred near 8 W/m2(irradiation time 10-12 s) (1, 3). The existence of the intensity threshold is especially important from a practical point of view (both in the laboratory and clinic) in deciding on the irradiation parameters. A similar type of curve was recorded at 454 nm for E. coli (10). Later, the invalidity of the reciprocity rule was shown for light at = 632.8 nm when irradiating human fibroblasts (18) and E. coli (19). It is clear that the same type of measurements are needed for all bands in action spectra. In the wavelength range used in our experiments, and important for phototherapy (600-860 nm), there are four “active” regions, but the peak positions are not exactly the same for all action spectra. The red band has a peak position between 613.5 and 623.5 nm (in one spectrum, at 606 nm); the far-red band has peak positions between 667.5 and 683.7 nm, and two near IR bands in the range of 750.7-772.3 nm and 812.5-846.0 nm.
3. INTERPRETATION of the ACTION SPECTRA: CYTOCHROME C OXIDASE is the PHOTOACCEPTOR MOLECULE In the beginning of the 90’s, the earlier action spectra (3-5, 17) were analyzed using all available spectroscopic literature data (9, 20), which allowed forming a suggestion about the chromophores involved. Bear in mind that the chromophores are the components of molecules that absorb the light. The action spectra for DNA and RNA synthesis rate (3-5), and changes in the adhesion of cells to a glass matrix (17) without Lorentzian curve fitting (which was not done at that time) were used for a
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summation, and this generalized action spectrum is shown in Figure 3.
Figure 3. The generalized action spectrum (a summation of 5 action spectra) for the increased proliferation of HeLa cells for = 330-860 nm (3-5,17). Curve 1: dose 10 J/m2, Curve 2: dose 100 J/m2. The suggested identity of the absorbing chromophores is marked, using data from (9). Figure 3 shows that the generalized action spectrum for cell proliferation in the range of 580-860 nm consists of two series of doublet bands in the range 620-680 nm and 760-895 nm with wellpronounced maxima at 620, 680, 760 and 825 nm. In the violet-blue region, there is one maximum at 400 nm, with the edge of the envelope near 450 nm. Recall that in the wavelength range 310-500 nm, a maximum stimulating effect was obtained with a radiation dose one order of magnitude less than in the longer-wave spectral range (3, 4). This is noted in Figure 3 by Curves 1 and 2. The bands in the action spectrum were identified in (20, and reviewed in 9) by analogy with the metal-ligand systems absorption spectra characteristic of this spectral range. The regions 400-450 nm and 620-680 nm are characterized by the bands pertaining to complexes with charge transfer in a metal-ligand system, and within 760-830 nm, these are d-d transitions in metals (21-23). The region 400-420 nm is typical of transitions in a porphyrin ring (24). Comparative analysis of spectral data for transition metals and their complexes on one hand, and biomolecules participating in the regulation of cellular metabolism on the other, allowed us to suggest that multinuclear enzymes containing Cu(II) may be participating (9, 20). Analysis of the electron excitation transitions of participating molecules containing Cu(II) (25-27) showed that metal-ligand transitions in the range of 400-450 nm correspond to the Nimidasole Cu transition, at 620 nm to the Scysteine Cu transition, and at 680 nm to the Smethionine Cu transition. Comparing the lines of possible d-d transitions and charge-transfer complexes of Cu (24-28) with Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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our action spectrum (Figure 3) allowed us to assume that the photoacceptor molecule has different types of centers containing Cu(II) in the ranges of 420-450 nm and 760-830 nm. In the range of 420450 nm, this may be a combination of centers of Types I and II (for the characteristics of centers of Types I, II, and III, see reference 23) though a center of Type I may be present. At 330 nm, a center of Type III may be present, and in the range 760-820 nm centers of Types I and III coexist. Within 620-680 nm, there is a center of Type I and a combination of centers of different types is unlikely. The above analysis allowed us to conclude that all bands in the action spectrum in Figure 3 may be related to cytochrome c oxidase (9, 20). The data of experiments of dichromatic irradiation and modification of light effects by adding various chemicals (1, 6-9) were taken into account as well in this analysis. In eukaryotic cells, cytochrome c oxidase is the terminal enzyme of the respiratory chain, which mediates the transfer of electrons from cyt c to molecular oxygen (29). Ferrocytochrome c is oxidized, oxygen is reduced, and protons are pumped from the mitochondrial matrix to the cytosol. Free energy resulting from this redox chemistry is converted into an electrochemical potential across the inner membrane of the mitochondrion, which ultimately drives the production of ATP. Accordingly, cytochrome c oxidase plays a central role in the bioenergetics of the cell. The respiratory chains of eukaryotic cells are located in mitochondria. Cytochrome c oxidase of mammalian cells is a large multicomponent membrane protein of considerable structural complexity. The high-resolution three-dimensional X-ray structure of cytochrome c oxidase of bovine heart (30-32) and Paracoccus denitrificans (33) were reported in 1995. These studies indicated that CuA is a binuclear copper center with an unexpected structure similar to a (2Fe-2S) type iron-sulfur center, in which the Fe ions and inorganic sulfur atoms are replaced with Cu ions and cysteine sulfur atoms, respectively. The O2 binding site contains heme a3 iron and CuB; there is no detectable bridging ligand between iron and copper atoms. Heme a is coordinated with two imidazoles of histidine residues. The fifth ligand of heme a3 is an imidazole, whereas CuB is coordinated by three imidazoles of histidine. Residues of two cysteins, two histidines, one methionine and one peptide carbonyl of a glutamate coordinate CuA (Cu-Cu) center (30). These reports of the crystal structures have opened a new era in cytochrome c oxidase research (34).
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Figure 4. The schematic of intramolecular electron transfer into the redox-active reaction center a3CuB of cytochrome c oxidase. Adapted from (31). Electrons are transferred sequentially from water-soluble cytochrome c to the CuA of cytochrome c oxidase, then to heme a, and to the binuclear center a3-CuB (catalytic center of cytochrome c oxidase), where oxygen is reduced to water (Figure 4). Oxygen binds to the catalytic center, and is reduced to water through a series of short-lived elusive intermediates. Singular value decomposition analysis indicated the presence of at least seven intermediates (35). The best-characterized species are the ferrous-oxycomplex and peroxy species (36-38). Generally speaking, cytochrome c oxidase can be fully oxidized (four redox active metal centers: CuA, CuB, irons in hemes a and a3, are in their common higher oxidation state; 3+ for iron and 2+ for copper), or fully reduced (four metal centers are in their common lower oxidation state; 2+ for iron and 1+ for copper). The partially reduced enzyme, usually called mixed-valence one, has some metal centers in their higher oxidation state, and the remainder in their lower oxidation state. There are also a number of forms of oxidized enzyme: fast enzyme (reacts relatively rapidly with cyanide), slow enzyme (reacts at about of 1% of the rate of the fast enzyme, also called resting enzyme), pulsed enzyme (obtained by reducing slow enzyme and oxidizing it with oxygen under conditions in which the production of H2O2 is avoided), oxygenated enzyme (subjected to a cycle of reduction and reoxidation under conditions in which H2O2 is produced) (38-40). These details are given to illustrate how complicated and controversial the overall picture of the function of cytochrome c oxidase still is. Coming back to the comparative analysis of the action spectrum in Figure 2, and the spectroscopic data on cytochrome c oxidase cited above, it was suggested (9, 20) that the 820 nm band belongs mainly to oxidized CuA, the 760 nm band to reduced CuB, the 680 nm band to oxidized CuB, and the 620 band to reduced CuA (Figure 3). The 400-450 nm band is more likely to be the envelope of a few absorption bands in the range 350-500 nm (i.e., a superposition of several bands). The band with a maximum near 404-420 nm can be assigned to the oxidized heme, whereas the longer-wave edge of the envelope at 450 nm (due to its asymmetry), should evidently be assigned to the reduced CuB. The participation of the heme in the action spectra is confirmed by the optimal dose ratio (10 and Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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100 J/m2, respectively, for 404 and other visible region maxima, (3-5)). It should be noted that the Soret band of heme compounds (i.e., the band in the range of 400-420 nm) is more intense by an order of magnitude than the absorption bands of these compounds in the visible region (24). The weak band at 330 nm may belong to oxidized CuB. Thus, the bands at 330, 404-420, 680 and 825 nm can be attributed to a relatively oxidized form of cytochrome c oxidase; the edge of the blueviolet band at 450 nm and the distinct bands at 620 and 760 nm belong to a relatively reduced form of the enzyme. This analysis (9, 20) was performed before the three-dimensional X-ray structure of cytochrome c oxidase was reported (30-33). The results of our analysis do not have principal contradictions with data on the structure of cytochrome c oxidase by Tsukihara et al. (30, 31). It should be emphasized that every band in the absorption spectra is a result of the overlapping absorption of different chromophores, but in an action spectra not all of them may appear. An analysis of the band shapes in the action spectra (Figure 3) and the line intensity ratios enabled us to conclude that cytochrome c oxidase cannot be considered as a primary photoreceptor when it is fully oxidized or fully reduced, but only when it is in one of the intermediate forms (i.e., partially reduced, or mixed valence enzyme) (9, 20). Our suggestion that cytochrome c oxidase is the photoacceptor responsible for various cellular responses connected with light therapy in the red-to-near IR region (9) was later conformed by the work of Pastore et al. (41), Wong-Riley et al. (11, 42), and Eells et al. (43, 44), as well as by our own spectroscopic work described below in the next Section (45, 46). A number of other kinds of experiments (dose and intensity dependences for various wavelengths, dichromatic irradiation in various ways, modification of irradiation experiments by specific chemicals, and others) were performed (reviews: 1, 6, 9, 47, 48). The results of all these experiments, together with action spectroscopy experiments, which were summarized briefly above, allowed the conclusion that cytochrome c oxidase could be a universal photoacceptor for eukaryotic cells.
4. COMPARISON of ACTION and ABSORPTION SPECTRA Insofar as an action spectrum mirrors the absorption spectrum of the molecule that absorbs the light and is responsible for the action spectrum recorded, an important step in identification of this photoabsorbing molecule is the comparison of action and absorption spectra. Recording an absorption spectrum of a cellular monolayer or individual cell is not an easy task, due to weak absorption and low concentration of chromophores. The absorption spectra of individual cells were recorded years ago up to 700 nm with the aim of identifying respiratory chain carriers (49, 50). The absorption spectrum of 8 parallel monolayers of human fibroblasts in the red-to-near IR region was recorded using a commercial double beam spectrophotometer (51). The absorption spectrum of whole cells in the visible region was found to be qualitatively similar to that of isolated mitochondria (49). The extension of optical measurements from the visible spectral range to the farred and near IR regions (650-1000 nm) was undertaken late in the seventies for the purpose of monitoring the redox behavior of cytochrome c oxidase in vivo. These studies led to the discovery of a “near IR window” into the body, and the development of near IR spectroscopy for monitoring tissue oxygenation (52-54). For recording the absorption of one cellular layer with the aim of studying the radiation-induced changes in the absorption of cell chromophores, a multichannel registration method was developed
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(55, 56). Figure 5 presents the absorption spectra (A-C), and the same spectra after the irradiation at 830 nm (A1-C1), as well as two action spectra (D, E) for comparison.
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Figure 5. Absorption spectra of HeLa cell monolayer: (A-C) prior to and (A1-C1) after irradiation at 830 nm. A, A1 (closed cuvette), B, B1 (open cuvette), C, C1 (air-dry monolayer). Original spectrum, curve fitting (solid lines) and Lorentzian fitting (dashed lines) are shown (adapted from (57)). Action spectra for: (D) stimulation of DNA synthesis and (E) stimulation of HeLa cell adhesion to a glass matrix, measured respectively 1.5 h after irradiation of HeLa cell monolayer (D= 100 J/m2, t = 10 s, I = 10 W/m2) and 30 min after irradiation of HeLa cell suspension (D = 52 J/m2, = 40 s, I= 1.3 W/m2). Experimental curves ( , adapted from (4, 5, 17)), curve fitting (solid lines) and Lorentzian fitting (dashed lines) are shown as described in (16). The cellular monolayers for all of our experiments were grown in closed cuvettes. The first series of experiments on the recording of the absorption spectra prior to and after irradiation were conducted with the cuvettes remaining closed. A typical spectrum recorded under these conditions prior to irradiation is presented in Figure 5A, and that recorded after irradiation, in Figure 5A1. Spectrum A is characterized by a strong absorption in the region of 730-850 nm (the bands resolved by the Lorentzian curve fitting method are at 736, 754, 773, and 797 nm, Table 3), and low absorption near 600 nm (a single band at 630 nm) and above 800 nm (the peaks at 830 and 874 nm are resolved, Table 3). This is the spectrum of the cells with the most strongly reduced cytochrome c oxidase in our experiments. The irradiation of the cells causes the following changes in the peak positions in this spectrum, as evident from spectrum A1 presented in Figure 3 and in Table 3. The low-height band is resolved in the red region at 634 nm. A single strong peak is resolved at 756 nm. The three bands with the peak positions at 807, 834, and 867 nm characterize the region of wavelengths over 800 nm. As a whole, both of these spectra, A and A1, have dominating bands in the region of 750770 nm. A typical spectrum of the cells with the most strongly oxidized cytochrome c oxidase in our experiments is presented in Figure 5C. In this case, the cuvettes were opened for 30 min to let the cellular monolayer dry in the air. The red band in this spectrum is resolved at 616 nm, and the farred bands, at 665 and 681 nm. The NIR region is characterized by weak bands at 712, 730 and 762 nm, and by two strong bands at 813 and 872 nm (Table 3). The irradiation of this monolayer caused no changes in the absorption spectrum, as shown in Figure 5 (spectrum C1) and Table 3. This is not an unexpected finding, because the respiratory chains do not function in dry cells. This experiment was needed to record the spectrum of the cells with oxidized cytochrome c oxidase. As a whole, spectrum C in Figure 5 was characterized by strong absorption bands in the red region, as well as in the NIR region of wavelengths over 800 nm, and extremely weak bands in the region of 750-770 nm. A comparison between spectra A and C allows us to conclude that the band at 750-770 nm is characteristic of relatively reduced photoacceptor molecules, and the band at 650-680 nm, as well as that at 800-870 nm, of oxidized ones. In the third series of experiments, the cuvettes were held open for 5 min prior to taking measurements. The absorption spectra of the cells in these conditions are typically characterized by a band at 633 nm in the red region, and the bands with the peak position at 666, 711, 730, 767, 791, and 880 nm in the far-red and NIR regions (Figure 5, Table 3). The irradiation of these cells causes significant changes in the absorption spectra in all spectral regions (spectrum B1 in Figure 5). The band at 750-800 nm becomes dominant in the spectra of the irradiated cells. There are no bands resolved at wavelengths over 800 nm, as well as in the region of 600-630 nm. Spectrum B1 features bands with the peak positions at 661, 681, 739, 765, and 788 nm (Table 3).
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Therefore, we performed a set of experiments to measure the absorption spectra of cultured cells the in region ~600-860 nm, and compared these spectra with action spectra recorded already. Then, we had to check if there were any changes in the absorption spectra due to the irradiation.
For quantitative characterization purposes, as well as for comparison between the recorded absorption spectra, we decided to use intensity ratios between certain absorption bands. The use of spectral band intensity ratios to analyze various spectra is not a new issue for spectroscopy in general (58), but to our knowledge, it has not been used in the absorption spectroscopy of isolated mitochondria or living cells. The calculation of the intensity ratios was shown to provide benefits for the exact comparison of spectra in the IR region (58). We used the band present in all absorption spectra near 760 nm (exactly at 754, 756, 767, 765, and 762 nm) (Table 3) as a characteristic band for the relatively reduced photoacceptor. The band used by us to characterize the relatively oxidized photoacceptor was the one near 665 nm (exactly at 666, 661, and 665 nm) in spectra B, B1, C, C1 (Table 3). This band is so weak that it could not be resolved by the Lorentzian fitting method in spectra A and A1, belonging to the most strongly reduced photoacceptor in our experiments. For this reason, we used in our intensity calculations for spectra A and A1 absorption on the curve fitting level at 665 nm. The gray vertical lines in Figure 5 mark the bands chosen. The intensity ratio I760/I665 was calculated to characterize every spectrum. In these simple calculations, we used only the peak intensities (peak heights), and not the integral intensities (peak areas) that are certainly needed for further developments. In the case of equal concentrations of the reduced and oxidized Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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forms of the photoacceptor molecule, the ratio I760/I665 should be equal to unity. When the reduced forms prevail, the ratio I760/I665 is greater than unity, and it is less than unity in cases where the oxidized forms dominate. Recall that the internal electron transfer within the cytochrome c oxidase molecule causes the reduction of molecular oxygen via several transient intermediates of various redox states (38, 59-61). The magnitude of the I760/I665 criterion is 9.5 for spectrum A (Figure 5), 1.0 for spectrum B, and 0.36 for spectrum C. By this criterion, irradiation of the cells, whose spectrum is marked by A (I760/I665 = 9.5) causes the reduction of the absorbing molecule (I760/I665 for spectrum A1 is equal to 16). Irradiation of the cells characterized by spectrum B also causes the reduction of the photoacceptor, as evidenced by the increase of the I760/I665 ratio from 1.0 to 2.5 in spectrum B1. In the spectrum of the cells with initially more reduced photoacceptor (spectrum A), irradiation causes reduction to a lesser extent (16/9.5 = 1.7) than in that of the cells with initially less reduced photoacceptor (spectrum B). The intensity ratio in this case is 2.5/1 = 2.5). Figure 5 also presents two action spectra, one for the stimulation of DNA synthesis in our HeLa cells (D), and the other for the stimulation of the attachment of the HeLa cells to a glass matrix (E). A comparison of peak positions in the absorption spectra with these two action spectra (Table 3) shows a similarity between them. The oxidized form of cytochrome c oxidase has a broad absorption band above 800 nm that is centered at 830 nm (62). CuA, a dimeric copper complex with four ligands, is responsible for 77% of the absorbency at 810-820 nm, while the contribution of heme a and heme a3/CuB is 18 and 5%, respectively (59). Due to the domination of the strong absorption of CuA in this region, weak underlying lines are masked in the absorption spectrum of cytochrome c oxidase (63). A distance of 33.5 nm between the minimal and maximal peak positions in the spectra (Table 1) is too long to be explained by measurement error (e.g., FWHM of irradiating light 10 or 14 nm). Quite probably, different lines in the absorption spectra of intermediates of cytochrome c oxidase appear in the action spectra. This suggestion, however, requires further experimental proof. A study of the near-IR absorption spectra of membrane-bound cytochrome c oxidase at low temperature shows that there are overlapping traces covering the full wavelength range from 680 to 870 nm (64-66). Flash photolysis causes the formation of a mixed valence compound with a peak at 740-750 nm, which supposedly could belong to invisible copper (CuB) (67). A band at ~785 nm is present in fully reduced, unliganded, five coordinated ferrous heme a32+(68). The addition of carbon monoxide to the reduced enzyme causes a blue shift from 785 nm to 760 nm in the difference spectrum, and the photodissociation of CO results in a reversion of the band from 760 to 784 nm (63). In the wavelength range 670-680 nm, there are no absorption bands of cytochrome c oxidase intermediates recorded so far. A small absorption band belonging to an intermediate (compound A) has been recorded at ~660 nm (59). The appearance of the 655-nm absorption band suggests that CuB is oxidized and participating in a spin-coupled state (69). It is suggested that the 655-nm feature may arise from a charge transfer band of ferric high-spin heme a3, which is modulated by the redox state of CuB. The 655-nm band disappears as the binuclear center is reduced (70). Quite probably, this is one line in the absorption spectra of cytochrome c oxidase and/or its intermediates. It is well known that reduced cytochrome c oxidase has a peak at 605 nm, and this peak has been
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recorded both in mitochondria and whole cells (71). For membrane-bound cytochrome c oxidase, this peak can be red-shifted ~10 nm, as compared to solubilized enzyme (69). Some isospectral peroxy intermediates have a peak at ~607 nm in their absorption spectra (59). The absorption of this region is 75% due to low spin heme a, and 25% by high spin heme a3. However, a definite contribution of CuA at ~615 nm has been suggested as well (72). The same technique of absorption measurements described in (57) was used to study oxidation or reduction of the photoacceptor under irradiation at = 632.8 nm as a function of dose (46). The comparison of action spectra connected with reactions in the cellular nucleus, and the absorption spectra of cellular monolayers at (600-860 nm allow one to conclude that by peak positions, these two groups of spectra may belong to the same molecule.
5. MITOCHONDRIAL SIGNALING: HOW the LIGHT-GENERATED SIGNAL in MITOCHONDRIA can INFLUENCE CELLULAR METABOLISM We know from the action spectra that the DNA and RNA synthesis rate is influenced by irradiation (Section 2), and we know that the photoacceptor (tentatively cytochrome c oxidase) is located in mitochondria (Section 3). There is an important question left: how the signal generated by the light quanta in cytochrome c oxidase is transduced to the nucleus. The answer is that mitochondrial retrograde signaling quite probable is responsible for this. Interested readers are guided to a recent review (73), however, below is a short summary. Recent work has uncovered an impressive number of extra mitochondrial factors that regulate the expression of nuclear genes for mitochondrial proteins. However, relatively little is known about how mitochondria send signals to the nucleus, and how the nucleus controls the expression of individual genes. One pathway of communication in cells from mitochondria to the nucleus that influences many cellular activities under both normal and pathophysiological conditions is mitochondrial retrograde signaling (74, 75). This recently discovered signaling is an opposite signaling pathway to a common and well defined pathway transforming information from the nucleus and cytoplasm to the mitochondria. Mitochondrial retrograde signaling sends information back to the nucleus about changes in the functional state of the mitochondria. The existence of a cellular signaling pathway: mitochondria cytoplasm (plasma membrane cytoplasm) nucleus, was proposed in 1988 (8). The reason to suggest the existence of such a cellular signaling pathway (then named photosignal transduction and amplification chain) was simple. It appeared that the action spectra for the increase of DNA and RNA synthesis rate could be recorded when cultured cells are irradiated in the region from 300 to 860 nm. The nucleus does not have chromophores absorbing in this region. Secondly, the data gathered to date showed that photoacceptors are located in the respiratory chain. So, it was then logical to suppose the existence of cellular signaling cascades between organelles. In 2004, a novel mitochondrial-signaling pathway in mammalian cells activated by red and near IR radiation was discovered (76). It was shown by Schroeder et al. (77) that IR-A radiation (760-1440 nm), in contrast to UV radiation, elicits a retrograde signaling response in normal human skin fibroblasts. Figure 6 presents a putative schematic of mitochondrial retrograde signaling activated by radiation in the visible and IR-A regions. This schematic was first proposed in 1988 (8, review: 47) and later supplemented with new details according to new experimental data (1, 9). Some new modifications
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are also included in the present schematic in Figure 6. Mitochondrial retrograde signaling was initially defined by an altered mitochondrial membrane potential (74). Later, other characteristics like changes in the concentration of mitochondrial ROS, Ca2+, and nitric oxide, as well as changes in fission-fusion homeostasis of mitochondria (75) were introduced. Changes in these characteristics under the irradiation with light at different wavelengths follow a cyclic pattern. First, a stimulation characterized by a threshold and phase of increase occurs. After a strict maximum and a phase of decrease, the control level is reached. As an example, at the level of a single cell and during real-time recording, a maximal increase in (30% of its basal value) was observed at 2 min after a 15 s mitochondrial membrane potential irradiation at 647 nm. Then decreased gradually to the basal level, which was reached 4 min later (78). Experimental data about the modulation of elements of mitochondrial retrograde signaling by irradiation are reviewed in (73). Irradiation of mammalian cells causes an upregulation of various genes (78). The upregulation of genes, and the increase in DNA and RNA synthesis rate (Sections 1, 2) are marked in Figure 6 in the nucleus of the cell. The cDNA microarray technique was used for human fibroblasts irradiated at 628 nm (79). Of the 9982 gene expression profiles studied, 111 genes in 10 function categories were upregulated. Note that among these 10 function categories, 7 of them were directly or indirectly involved in cell proliferation. The other 3 function categories upregulated were genes related to transcription factors, immune/inflammation, and cytokines as well as some genes not identified (79). It should be noted that the responses of mammalian cells to visible and near IR radiation, as well as the sensitivity of the mitochondrial respiratory chain components to this radiation have never gained as much serious attention by photobiologists, as have the functional photoacceptors, such as chlorophyll and rhodopsin. However, fragmentary knowledge gathered so far forces one to ask whether the photosensitivity of some enzymes of the mitochondrial respiratory chain may have a physiological significance in spite of the complete adaptation of living systems to photons as a natural external factor.
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There is every reason to believe, on the basis of experimental data gathered so far, that mitochondrial retrograde signaling, a recently discovered cellular signaling pathway, functions also in irradiated cells. Modulation of retrograde mitochondrial signaling elements like , (ROS)m, (Ca2+)m in irradiated cells is rather well documented (review: 73). Also, the responses to irradiation occurring in the nucleus (i.e., increase in DNA and RNA synthesis rate, and expression of genes of various function categories) are definitely documented. However, the pathways of light signal transduction between these two ends needs further investigation.
6. CONCLUSIONS 1. The similarity of action spectra for different cellular responses suggests that the photoacceptor is the same for these responses. For the responses reported here, the photoacceptor appears to be cytochrome c oxidase. Recall that it was suggested in 1981 that photosensitivity might be a common mitochondrial property in higher animals, and could have physiological significance under certain conditions, e.g., exposure to orange-red light, and high ADP levels (80). 2. Based on these action spectra, various wavelengths can be used for low level light therapy, i.e., those around 404, 620, 680, 760, and 820 nm.
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3. The existence of the action spectra for biochemical processes occurring in various cellular organelles (nucleus, plasma membrane) assume the existence of cellular signaling pathways between a photoacceptor in the mitochondria and the nucleus, as well as between photoacceptor and the plasma membrane. 4. It is believed that the “mitochondrial mechanism” of low level light therapy works in all types of cells containing mitochondria (1).
Naeser Laser Home Treatment Program for the HAND Carpal Tunnel Syndrome Pain or Hand Paresis in Stroke Treated with Laser Acupuncture and Microamps TENS –
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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Teaching Module Abstract presented at the 3rd Annual Meeting of the North American Association for Laser Therapy (NAALT), Uniformed Services University for the Health Sciences, Bethesda, MD, April 4, 2003. www.naalt.org
Margaret A. Naeser, Ph.D., Lic.Ac., Department of Neurology Boston University School of Medicine and V.A. Boston Healthcare System. Psychology Research (116-B), VA Boston Healthcare System, 150 So. Huntington Ave., Boston, MA 02130
[email protected] This program may be used with Carpal Tunnel Syndrome (CTS). It may also be used to treat mild-moderate hand paresis (weak, clumsy hand) in stroke patients or other CNS disorders (head injury, encephalitis, spinal cord injury or M.S.). Patients with hand paresis may have some improved hand function after 3 months of treatment. If there is severe hand spasticity (“fisted” hand in flexion) there may be no improved hand function following this treatment program, however, there may be some reduction in the spasticity following at least 3 months of treatment. This program may also be used with Raynaud's, or peripheral neuropathy in the hands (earlier stages), rheumatoid or osteoarthritis (earlier stages). Treat 3-6 times per week. For CTS cases, treat every 48 hours, for 5 weeks. This protocol includes both microamps TENS and red-beam low-level laser. For CTS cases, infra-red laser is also recommended on deeper acupuncture points at cervical paraspinal areas C5 - C8, T1; and shoulder, elbow and forearm areas where radiating pain may be present. No medical claims are made. Some lasers which have been used include: Red-beam Lasers (for shallow acupuncture points). Lasotronic Pocket Therapy Laser, 45 mW, 660 nm, CW. Elliptical beam, 1.5 mm x 3.5 mm. Beam spot size: 0.0412 cm2. 1 Joule/cm2 = .915 sec. or 1 sec. 4 Joules/cm2 = 4 sec. Ito Laser Pointer, 5mW, 670 nm. CW. Aperture: 5 mm diameter. Beam spot size: 0.196 cm2 1 Joule = 200 sec. 1 Joule/cm2 = 39.2 sec. 4 Joules/cm2 = 156.9 sec. or 2 Min. 36 sec. Infrared Lasers (for deeper acupuncture points). Respond 2400XL Laser (Now, Luminex), 500 mW, 904 nm, CW. Aperture: 1.14 cm diam. Beam spot size: 1.03 cm2 1 Joule = 0.5 sec. 1 Joule/cm2 = 2 sec. 4 Joules/cm2 = 8 sec
Microamps TENS Device: MicroStim 100 TENS (580 µA to 3.5 mA) with 2 circular electrodes, where each electrode has a copper-coated surface, with four embedded, tiny, red LEDs (not laser light). MicroSti m 100 TENS Devi ce pacemaker.)
(TENS may not be used in pregnancy, nor with a
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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1. Be sure the hand and fingers of the patient are clean and dry; no perspiration or hand lotion, etc. Wipe the areas where the acupuncture points are located with an alcohol pad. See attached Diagram. 2. Set switch to the "square wave" on the MicroStim 100 TENS device. 3. There are 2 circular electrodes, each has a copper-coated surface, with four embedded, tiny red LED lights. Place one double-sided sticky CLEAR circular conducting patch onto the copper surface of each circular electrode. 4. For CTS cases, PC 7 is first treated at an energy density of 32 Joules/cm2 with red-beam laser - see #1 below, under Low-Level Laser Treatment. For CTS cases, after PC 7 has been treated with the laser, place one circular electrode with attached sticky patch over PC 7 and place the second circular electrode over TW 4 on the dorsum of the wrist, so that the two circular electrodes are placed opposite to each other (TW 4 and PC 7), to enable treatment through the wrist with the MicroStim 100 TENS. (Ignore grounding pad shown on diagram, use circular electrode, instead.) For stroke patients, place one circular electrode with attached sticky patch, onto the palm of the hand, so that at least a portion of it COVERS acupuncture points Hrt 8 and PC 8. Place the second circular electrode over LI 4 or TW 5. 5. Set the frequency switch on the top of the MicroStim 100 TENS to F4 (292 Hz) for the first frequency to use. 6. Turn the round power control knob to "On," and SLOWLY turn up the power until the patient reports feeling a "tingling sensation" from either electrode which is in place. Now, turn the power down, until the PATIENT DOES NOT FEEL ANY STIMULUS AT ALL. This will be the correct setting (it should be around only 2 or 4 on the round power control knob). If the patient does not report any tingling sensation at all, even at the highest setting of 9, this is OK and just set the power to maximum. Leave the power setting at this subthreshold level, for 2 minutes. 7. After the first 2 minutes at F4, switch the frequency knob over to the lowest frequency setting of F1 (0.3 Hz) for 18 minutes. The MicroStim 100 TENS device turns itself off after 20 minutes. Discard both circular sticky patches; do not store the used patches on the LED surface, this would corrode the copper; do not re-use the sticky patches after one use. The total time required for the MicroStim 100 TENS treatment is 20 minutes.
Low-Level Laser Treatment While the microamps TENS treatment is ongoing, a red-beam laser can be used to treat the JingWell points near the base of the fingernail beds, and other points on the hand, at an energy density of 4 J/cm2. The tip of the laser pointer should physically touch the skin, but do not press so hard that the tip leaves a very deep indentation on the skin. Hold the laser pointer at a right angle to the skin. These acupuncture points include: 1. If CTS is being treated, treat acupuncture point PC 7 (closest point to the median nerve at the wrist crease) with red-beam laser BEFORE placing the circular electrode of the TENS device on PC 7. Use 32 J/cm2 at PC 7 (for Lasotronic 45 mW Laser, 32J/cm2 = 32 sec.; for ITO 5 mW laser, 32J/cm2 = 21 minutes). After this treatment with the laser, place the TENS electrodes on PC 7 and TW 4 to treat through the wrist, as explained in #4-#7, above. 2. While the TENS is in place, use the laser to treat the following acupuncture points: Lu 11, LI 1, PC 9, TW 1, Hrt 9, SI 1. See attached Diagram. These are important points. Each point is
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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treated at 4 J/cm2 (for Lasotronic 45 mW laser, 4 J/cm2 = 4 sec.; for ITO 5 mW laser, 4 J/cm2 = 2 min. 36 sec.). These points are treated for all disorders, including CTS. 3. Lu 9, Hrt 7. These points located at the wrist crease, are important for CTS cases. See Diagram. 4. Optional points for severe finger cases (e.g., arthritis): Extra points at ends of major finger creases at the joints, on radial and ulnar side of fingers. These are the proximal and distal interphalangeal joints. There are 4 of these extra points on each finger, and 2 on each thumb. Each point is treated, 4 to 8 J/cm2. 5. Optional points are distal Ba Xie points in webspaces btwn. fingers; or proximal Ba Xie points. 6. Other hand points, LI 4 or TW 3. Use the red-beam laser to treat shallow points, on adults. 7. It is recommended that acupuncture needles or infrared laser be used to treat deeper points on adults, such as LI 11, LI 15, TW 9, and with CTS cases, the cervical paraspinal areas are treated lateral to C5 - C8 and T1 (Hwa To points). These points are treated at 4 - 8 J/cm2 (for Respond 2400XL 500mW laser, 4 - 8 J/cm2 = 8 - 16 sec. per point). If there are tender points on palpation (especially on the forearm), treat for 4 - 8 J/cm2, and then re-palpate that point/area, and treat again, until there is a change in the sensitivity level there. When used with CTS patients, there was a success rate of 88% (Naeser, Hahn, Lieberman, Branco, 2002; controlled study) to 92% (Branco & Naeser, 1989; open protocol) where success was defined as a reduction of at least 50% in the pain level. Eligible CTS patients: NCS motor latency should not be greater than 7.0 msec (4.3 msec = WNL). For best results, treat 3 times per week for 5 weeks. Treatment results were stable at 1 – 6 year follow-up in 90% of the patients.
References Branco K & Naeser MA: Carpal tunnel syndrome: Clinical outcome after low-level laser acupuncture, microamps transcutaneous electrical nerve stimulation, and other alternative therapies - an open protocol study. The Journal of Alternative and Complementary Medicine, 5(1): 5-26, 1999. Naeser MA, Hahn K-A K, Lieberman BE, Branco KF. Carpal Tunnel Syndrome Pain Treated with LowLevel Laser and Microamps TENS, A Controlled Study. Archives of Physical Medicine and Rehabilitation, 2002;83:978-988. Naeser MA. Photobiomodulation of Pain in Carpal Tunnel Syndrome: Review of Seven Laser Therapy Studies. Photomedicine and Laser Surgery, 2006; 24(2):101-110.
Naeser MA, Wei XB, Laser Acupuncture - Introductory Textbook for Treatment of Pain, Paralysis, Spasticity and Other Disorders, Boston, Boston Chinese Medicine, 1994, p. 40. Available througth Lhasa OMS, www.LhasaOMS.com or 1-800-722-8775. Also through
[email protected] Websites: www.bu.edu/naeser/acupuncture http://gancao.net/ht/cts.shtml http://gancao.net/ht/laser.shtml
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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Sp 1
Liv 1
St 45 GB 44
Right Foot 4 Joules/cm 2 on each acupuncture point
Bl 67
Circular Electrode Liv 3
The second circular electrode is placed on the sole of the foot opposite to Liv 3 (near Ki 1).
(For the left foot, the point locations are anatomically identical.)
Naeser Laser FOOT Treatment Program
Naeser Laser Home Treatment Program for the FOOT Peripheral Neuropathy and Poor Circulation in the Foot Treated with Laser Acupuncture and Microamps TENS Teaching Module Abstract presented at the 3rd Annual Meeting of the North American Association for Laser Therapy (NAALT), Uniformed Services University for the Health Sciences, Bethesda, MD, April 4, 2003. www.naalt.org
Margaret A. Naeser, Ph.D., Lic.Ac., Department of Neurology Boston University School of Medicine and V.A. Boston Healthcare System. Psychology Research (116-B), VA Boston Healthcare System, 150 So. Huntington Ave., Boston, MA 02130 email:
[email protected] May be used with peripheral neuropathy (of diabetic, AIDS, or neurological origin) or poor circulation to the feet, including foot ulcers. (AIDS patients taking certain medications can develop serious, painful, peripheral neuropathies in the ankles/feet.) This protocol could also be tried with stroke patients or M.S. patients with mild ankle dorsi-flexion problems ("foot drop"). It can also be used with spinal cord injury patients who have cramping and spasticity in the leg and foot muscles. Treat 3-6 times per week. This protocol includes both microamps TENS and red-beam low-level laser. No medical claims are made.
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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Some lasers which have been used include: Red-beam Lasers (for shallow acupuncture points). Lasotronic Pocket Therapy Laser, 45 mW, 660 nm, CW. Elliptical beam, 1.5 mm x 3.5 mm. Beam spot size: 0.0412 cm2. 1 Joule/cm2 = .915 sec. or 1 sec. 4 Joules/cm2 = 4 sec. Ito Laser Pointer, 5mW, 670 nm. CW. Aperture: 5 mm diameter. Beam spot size: 0.196 cm2 1 Joule = 200 sec. 1 Joule/cm2 = 39.2 sec. 4 Joules/cm2 = 156.9 sec. or 2 Min. 36 sec. Infrared Lasers (for deeper acupuncture points). Respond 2400XL Laser (Now, Luminex), 500 mW, 904 nm, CW. Aperture: 1.14 cm diam. Beam spot size: 1.03 cm2 1 Joule = 0.5 sec. 1 Joule/cm2 = 2 sec. 4 Joules/cm2 = 8 sec Microamps TENS Device: MicroStim 100 TENS (580 µA to 3.5 mA) with 2 circular electrodes, where each electrode has a copper-coated surface, with four embedded, tiny, red LEDs (not laser light). MicroSti m 100 TENS Dev ice (A TENS device may not be used with a patient who is pregnant, nor with a patient who has a pacemaker.) 1. Be sure the foot and toes of the patient are clean and dry; no perspiration or body lotion, etc. Wipe the areas where the acupuncture points are located with an alcohol pad. See Diagram on last page. 2. Set switch to the "square wave" on the MicroStim 100 TENS device. 3. There are 2 circular electrodes, each has a copper-coated surface, with four embedded, tiny red LED lights. Place one double-sided sticky CLEAR circular conducting patch onto the copper surface of each circular electrode. 4. Place one circular electrode with attached sticky patch, onto the top of the foot at Liv 3. Place the second circular electrode with attached sticky patch on the sole of the foot at Ki 1. See Diagram. Place this second circular electrode so that the two circular electrodes are placed opposite to each other, to enable treatment through the foot with the MicroStim 100 TENS device. 5. Set the frequency switch on the top of the MicroStim 100 TENS to F4 (292 Hz) for the first frequency to use. 6. Turn the round power control knob to "On," and SLOWLY turn up the power until the patient reports feeling a "tingling sensation" from either electrode which is in place. Now, turn the power down, until the PATIENT DOES NOT FEEL ANY STIMULUS AT ALL. This will be the correct setting (it should be around only 2 or 4 on the round power control knob). If the patient does not report any tingling sensation at all, even at the highest setting of 9, this is OK and just set the power to maximum. Leave the power setting at this subthreshold level, for 2 minutes. 7. After the first 2 minutes at F4, switch the frequency knob over to the lowest frequency setting of F1 (0.3 Hz) for 18 minutes. The MicroStim 100 TENS device turns itself off after 20 minutes. Discard both circular sticky patches; do not store the used patches on the LED surface, this would corrode the copper; do not re-use the sticky patches after one use. The total time required for the MicroStim 100 TENS treatment is 20 minutes. Low-Level Laser Treatment
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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While the microamps TENS treatment is ongoing, a red-beam laser can be used to treat the JingWell points near the base of the toenail beds, and other points on the foot, at an energy density of 4 J/cm2. The tip of the laser pointer should physically touch the skin, but do not press so hard that the tip leaves a very deep indentation on the skin. Hold the laser pointer at a right angle to the skin. These acupuncture points include: 1. Sp 1, Liv 1, St 45, GB 44, Bl 67. These are the most important points; others are optional. See Diagram on last page. 2. Points in the web spaces between the toes. 3. Ki 6, Sp 5, Liv 4, St 41, GB 40; Ki 3, Bl 60; and/or other shallow ankle and foot points. These points are optional. They are located around the ankle. See an acupuncture textbook for their locations. 4. Use the red-beam laser only on shallow points, on adults. 5. The 5 mW red-beam laser may be used at increased J/cm2 (8 J/cm2), as necessary, on very painful joints/areas. 6. It is, of course, recommended that acupuncture needles or infrared laser also be used to treat other deeper acupuncture points on the leg, as appropriate, on adults. 7. The patient can perform this treatment on him/herself once a day, or every other day, until there is stable pain relief (or reduced spasticity over several days); then the treatments can be used only as necessary. 8. For cases with leg cramping and spasticity (especially spinal cord injury cases), also consider the use of a magnet cap developed by Agatha Colbert, M.D. The magnet cap appears to affect GV 20 and SiShenCong, reducing spasticity in the legs and reducing stomach spasms in spinal cord injury patients (Naeser, personal observation). The magnet cap may be purchased through Jayne Ronsicki, Lic.Ac., Hudson, MA.
[email protected] 1-877-527-1550 or 978-562-6389. Steve Liu, Lic.Ac., Tucson, AZ has experience using this (or a similar) FOOT treatment program combining red-beam laser on acupuncture points and microamps TENS, in treatment of diabetic peripheral neuropathy (Liu, 2002 and personal communication).
[email protected] References Liu, S. 2002. Treatment of Diabetic Peripheral Neuropathy with Low Power Laser Acupuncture. Poster presentation at the Society for Acupuncture Research Meetings, Seattle, Washington, October, 2002. Naeser MA, Wei XB, Laser Acupuncture - Introductory Textbook for Treatment of Pain, Paralysis, Spasticity and Other Disorders, Boston, Boston Chinese Medicine, 1994, p. 41. Available througth Lhasa OMS, www.LhasaOMS.com or 1-800-722-8775. Also through
[email protected] Websites: www.bu.edu/naeser/acupuncture
http://gancao.net/ht/laser.shtml
Some sources for devices mentioned here: Lasotronic Pocket Therapy Laser, 45 mW, 660 nm.
[email protected] (Zurich, Switzerland) Ito Laser, 5mW, 670 nm, Lhasa Medical, Weymouth, MA 800-722-8775, www.LhasaOMS.com $118.00, replace two AAA batteries after three hours of use. If 2 lasers purchased, cost $112/laser. Luminex Laser (Formerly Respond Laser), 500 mW, 670nm, 867 nm, 904 nm. Medical Laser Systems, Branford, CT 800-778-0836,
[email protected] www.medicallasersystems.com Thor Lasers. Variety of laser cluster heads and single probes, red-beam and infrared.
[email protected] www.Thorlaser.com Phone: 540-942-4500 188 Sherwood Dr., Waynesboro, VA 22980
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MicroStim 100 TENS unit with 2 circular electrodes. MicroStim, Inc., Palm City, FL. 1-800-326-9119 or (954) 720-4383. Developed by Joel Rossen, DVM.
[email protected] [email protected] $295.00
Table 1. Acupuncture or Laser Acupuncture to Treat Paralysis in Stroke
Authors Naeser, Alexander, Stiassny-Eder, Galler, Hobbs, Bachman, 1992 Boston Univ. Sch. Med Boston VA Medical Ctr Naeser, Alexander, Stiassny-Eder, Galler, Hobbs, Bachman, 1994a
No. Cases Real Acupuncture 10 Acute Arm/Leg Cases, Starting at 1 - 3 Months poststroke, 20 Real Tx.’s, 4 Wks. 10 Acute 10 Chronic Arm/Leg Cases, Starting Acute: 1 - 3 Mo. Chronic:
Number Control Cases Sham or No Acptr. 6 Acute Arm/Leg Cases, 1 - 3 Mo.’s poststroke, 20 ShamTx.’s 4 Wks. Acute Arm/Leg Control Cases, see above study with
Significance Level between Groups and/or Number of Cases with Outcome Level of Good Response/Markedly Effective p < .013, with CT Scan Lesion Site as a Variable 4/10 Good Response, Real Acptr. 0/6 Good Response, Sham Acptr.
3 Chronic Arm/Leg Cases, No Acptr.
p < .003 (Chronic Cases), with CT Scan Lesion Site as a Variable 3/10 Good Response, Chronic Cases, Real Acptr. 0/3 Good Response, Chronic Cases, No Acptr. 5/10 Good Response, Acute Cases, Real Acptr. Isolated Active ROM for 8 Good Response Cases:
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Authors
No. Cases Real Acupuncture
Number Control Cases Sham or No Acptr.
Significance Level between Groups and/or Number of Cases with Outcome Level of Good Response/Markedly Effective
Boston Univ. Sch. Med Boston VA Medical Ctr
4 Mo. to 6 Yr. poststroke, 20 - 40 Tx.’s, 2 - 4 Months
Sham Acptr.
-20 Tx.’s p level -40 Tx.’s p level Shoulder Abd. +7 % < .04 +12 % < .04 Knee Flexion +19 % < .02 +22 % < .03 Knee Extens. +19 % n.s. +28 % < .01
Naeser, Alexander, Stiassny -Eder, Lannin, Bachman, 1994b Boston Univ. Sch. Med Boston VA Medical Ctr Johansson, Lindgren, Widner, Wiklund, Johansson, 1993 Lund Univ., Sweden Magnusson, Johansson, Johansson, 1994 Lund Univ., Sweden Sallstrom, Kjendahl, Osten, Stanghelle, Borchgrevink, 1995 Oslo, Norway Hu, Chung, Liu, et al., 1993
Taipei, Taiwan
3 Acute 8 Chronic Hand Cases Acute: 1 - 3 Mo. Chronic: 4 Mo. - 8 Yr., 20 - 40 Tx.’s 38 Acute Cases, 4 - 10 days poststroke, 20 Tx.’s (twice per week, 10 weeks) + P.T.
2 Chronic Cases, No Acptr.
p < .022 (Chronic Cases) All Acptr. Cases, Good Response, 11/11 = 100% 0/2 Good Response, Chronic Cases, No Acptr. Finger Strength Testing for 8 Chronic Acptr.Cases Tip Pinch: +3 lbs., -40 Tx.’s, p < .04 Palmar Pinch: +3 lbs., -20 Tx’s, p < .01
40 Acute Cases, 4-10 Days poststroke, P.T. Only
Savings of $26,000 per Acupuncture Patient due to reduced number of days in Rehab. Facilities p < .01 and beyond for: Walking and Balance at 1 Mo. and 3 Mo. Activities of Daily Living at 3 Mo. and 12 Mo. Quality Life, Mobility and Emotion at 3,6,12 Mo.
21 Acute Cases from above Johansson study
21 Acute Cases from above study
Follow-up on Postural Control 2 Yrs. later: p < .01, greater Postural Control for Cases Tx.’d with Acupuncture beginning 4 - 10 days poststroke
24 Subacute Cases, 40 days poststroke, 18 - 24 Tx.’s, 6 Weeks
21 Subacute Cases, 40 Days poststroke, P.T. Only 15 Acute Cases, No Acptr.
Cases who received Acupuncture were better after 6 weeks on the following: Motor Function, p = .002 Activities of Daily Living, p = .02 Quality Life, Nottingham Health Profile, p = .009
15 Acute Cases, Acupuncture Treatments started within 36 hours poststroke
Neurologic Outcome better at 1 Mo. p = .02, and at 3 Mo. p = .009 for Acute Cases Tx.’d with Acupuncture within 36 hours poststroke Results significant for Severe Subgroup at 1 Mo. p = .009, and at 3 Mo. p = .013; but not significant for Mild-Moderate Subgroup.
Table 1, Cont’d. Acupuncture or Laser Acupuncture to Treat Paralysis in Stroke
Authors Zhang, Li, Chen, Zhang, Wang, Fang, 1987
No. Cases Real Acupuncture
Number Control Cases Sham or No Acptr.
53 Acute and Chronic Cases, 24 Tx.’s,
41 Acute and Chronic
Significance Level between Groups and/or Number of Cases with Outcome Level of Good Response/Markedly Effective Acupuncture Group: 44/53 Cases, 83%, increased muscle strength by 1 - 2 grades at 6 joints: shoulder, elbow, wrist, hip, knee, ankle
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Authors
No. Cases Real Acupuncture
Number Control Cases Sham or No Acptr.
6 Tx.’s per Week, for 6 Weeks
Shanxi College Traditional Chinese Medicine, Shanxi, China
Naeser, Alexander, Stiassny-Eder, Galler, Hobbs, Bachman, Lannin, 1995
Boston Univ. Sch. Med Boston VA Medical Ctr
Cases No-Acupuncture Group: 26/41 Cases, 63% Difference between Groups: p < .05
Shanghai Medical Univ. China Li, Li, Wei, Zhao, Lu, 1989
Significance Level between Groups and/or Number of Cases with Outcome Level of Good Response/Markedly Effective
Acute Cerebral Hemorrhage, Two Groups Received Two Types of Acupuncture: Group 1 (n=46), Midline, base of skull, GV 16, GV 15, plus body points Group 2, (n=46), body points only 42 - 56 Tx.’s, daily
Laser Acupuncture 5 Arm/Leg Cases, 2 Hand Cases, (6 Chronic, 10 Mo. to 6.5 Yr. poststroke; and 1 Acute Case), 20 - 60 Tx.’s, over 2 - 4 Mo., 20 mW, 780 nm 1 Joule per point
Cases were treated within 24 hours, to a week, post-hemorrhage. Most bleeding completed within 4 hours in acute cerebral hemorrhage cases. Group 1: 38/46 Cases, 82.6%, Markedly Effective Group 2: 17/46 Cases, 37%, Markedly Effective Difference between Groups: p < .01 Acupuncture points GV 15 and GV 16 highly recommended in acute cerebral hemorrhage cases.
5/7 Cases (71%) Good Response Results similar to results with needle acupuncture where similar CT scan lesion sites were observed.
Overall, Good Response Post-Acupuncture, 128/193 Cases = 66.3%
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Table 2. Acupuncture/Laser Acupuncture to Treat Cerebral Palsy in Babies and Children No. Cases Number Control Significance Level between Groups and/or Number of Cases with Outcome Level of Real Cases Authors Acupuncture Sham or NoAcptr. Good Response/Markedly Effective Filipowicz, 1991
Warsaw, Poland Toronto, Canada Lao, 1992 New York City Shi, Bu, Lin, 1992 Shanghai Medical Univ. Shanghai, China Xiao & Meng, 1995
Beijing, China Ma & Zhang, 1995 Clinic of Lanzhou Brewery, Gansu, China Spears, 1979
Chatham, NJ Lidicka & Hegyi, 1991
Prague, Czech Republic Budapest, Humgary
65/65 Cases, 100%, Considerable Improvement
65 Babies and Children, age 40 days to 4 Years, Acupressure, Needle Acptr., Laser Acptr. (2-10 mW, redbeam Laser), Electroacptr., 2-3 Tx.’s per week, over a 5-Year period 10-month old baby, Needle Acptr., 50 Tx.’s, over a 5-Month period
4 Cases, “Complete Recovery” when Acptr. Tx.’s started at less than 6 months of age. The earlier the Acptr. Tx.’s initiated, the greater the reduction in spasticity. Laser Acptr. especially effective to treat contractures of Achilles tendon; after 30 - 60 seconds of exposure, “considerable and immediate improvement.” Pre-Acptr: Unable to sit up (with or without assistance); Achilles tendons tight, bilaterally Post-10 Acptr. Tx.’s: Able to sit, started to crawl, spasticity alleviated Post-50 Acptr. Tx.’s: At 15 months of age, walking independently, similar to children his age.
117 children, age 6 Mo. to 10 Yr., 30 Acptr. Tx.’s, 4 - 5 Months
63/117 Cases, 53.8%, Markedly Improved or Better
30 children, age 1 - 14 Yr., 30 Tx.’s, 66-day period Ear Stimulation + Limb Massge.
30 children 1 - 14 Yr., 30 Tx.’s, 66-days, Only Limb Massage
Ear Stimulation + Massage: 16/30 Cases, 53%, Improved Massage Only: 4/30 Cases, 13.3%, Improved
48 children, age 1 - 6 Yr.; and 12, over 6 Yr., Acptr. Tx.’s, 1 - 4 Months
9 children age 1-6 Yr, 3, >6 Yr. Vitamins and Herbs, 1-3 Month
Acptr. Group: 39/60 Cases, 65%, Markedly Improved Vitamins & Herbs Group: 2/12 Cases, 16.6%, Markedly Improved
5, teenage 1 child, 4.8 Yr., ElectroAcptr., Ear Stimulation At least 8 Tx.’s Laser Acupuncture 5 mW, redbeam 145 children, age 2 Wks. to 5 Yr., Tx’d for several Mo’s.-Yr.’s.
Difference between groups: p < .01
Difference between Groups: p < .01 6/6 Cases, 100%, Less spasticity, loosened Achilles tendon, control of drooling Majority of Cases, Less Spasticity, Improved Motor Function for Sitting, Crawling, and Walking Recommend Laser Acptr. Tx.’s be used with babies likely to develop cerebral palsy, starting at 2 weeks post birth; 2 years of age is considered late to begin Acptr. Tx.’s.
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
Overall, Good Response Post-Acupuncture, 190/279 Cases = 68.1%
Naeser Laser Home Treatment Program for Children with Cerebral Palsy and Motor Developmental Delay In an ideal situation when treating cerebral palsy, start treatment 2 weeks post-birth, using the 5 mW red-beam ITO laser pen, Continuous Wave, for only 5 - 10 seconds per acupuncture point (Lidicka & Hegyi, 1991).
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
For a 4-year old child, for example, use the 5mW red-beam ITO laser pen, Continuous Wave, for about 30 seconds per acupuncture point. The 4-year old child we worked with liked to practice reciting the alphabet (about 30 seconds) per acupuncture point, while the mother was treating a point with the laser. Treat 3 – 6 times per week. Try to treat for only 10 minutes. Treat for years. The pediatric Chinese Herbal Medicine formula, Liu Wei Di Huang Wang is often used with children with developmental delay. There are two basic sets of points (Extra Meridians), which are alternated between the treatments (Set A and Set B). Note the following, regarding the sequence of points to be treated: On a male child, treat the Master Point first, on the left side, only. On a male child, then treat the Coupled Point, on the right side, only. On a female child, treat the Master Point first, on the right side, only. On a female child, then treat the Coupled Point, on the left side, only. Set A (Governing Vessel) Master Point – SI 3; Coupled Point – BL 62 5
Set B (Dai Mai) Master Point – GB 41; Coupled Point – TW
Note: The Yang Chaio Mai is also helpful to increase strength and agility in the feet, used with Motor Developmental Delay. Master Point - BL 62; Coupled Point - SI 3.
General Points to also Treat Every Day or Every Other Day GV 15, and 16 (very important) ST 36 LI 11, and 4 LIV 3 BL 23, and 17 GB 41 and TW 5 are the most important points to treat for long-term use If child is agitated – LIV 3 and LI 4 (bi-lateral) If leg turns outward – SP 5, KI 2, KI 6 If wrist turns outward – PC 6
If leg turns inward – GB 40, BL 62 If wrist turns inward – TW 5
Ear Points: (Treat ear points for less time). Corpus Callosum, Hypothalamus, Laterality Point, L; Oscillation Point (R and L). Yamamoto New Scalp Needle Acupuncture Points: (Bilateral) Brain Point; Liver Point, temple area Other Points: BL 57 (for spasms) GB 34, and 39 ST 40 KI 3 GV 14 Dr. Michaela Lidicka, Prague, Czechoslovakia and Dr. Gabriella Hegyi, Budapest, Hungary. Papers presented at ICMART Meetings, Munich, Germany, June 14-17, 1991. Summarized in Naeser MA, Wei XB. Laser Acupuncture – An Introductory Textbook for Treatment of Pain, Paralysis, Spasticity and Other Disorders. Boston, Boston Chinese Medicine, 1994, p. 77.
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
Acupuncture and Laser Acupuncture to Reduce Symptom Severity in Spinal Cord Injury Margaret Naeser, Ph.D., Lic.Ac., Dipl.Ac. (NCCAOM) Neuroimaging/Aphasia Research, Boston V.A. Medical Center Department of Neurology, Boston University School of Medicine
[email protected] World Health Organization Conference: Review of Emerging Therapeutic Options for Human Spinal Cord Injury, Reykjavik, Iceland, June 1-2, 2001 The National Institutes of Health (NIH), Bethesda, MD held a Consensus Development Conference on Acupuncture in November, 1997. Portions of the material included here are taken from my invited report for that conference, “Neurological Rehabilitation: Acupuncture and Laser Acupuncture to Treat Paralysis in Stroke and Other Paralytic Conditions and Pain in Carpal Tunnel Syndrome.” The section "Other Paralytic Conditions" included Spinal Cord Injury. The conference was sponsored by the NIH Office of Alternative Medicine and the Office of Medical Applications of Research; the Proceedings were published by NIH in 1997 (pp. 93-109). The attached Table summarizes results from three studies performed in China before 1997, where acupuncture was used to treat Spinal Cord Injury (Gao, 1984; Gao et al., 1996; Wang, 1992). None had a control group. Overall, 340/360 cases, 94.4%, had an outcome level of beneficial progress, including reduction in muscle spasms, some increased level of sensation, improved bladder and bowel function. Patients were treated ranging from 5 months to 2-3 years, or even for 5 years. Electroacupuncture along the Bladder meridian (paravertebral) area was especially recommended. Authors recommend beginning acupuncture as soon as possible after spinal cord injury, even during the acute stage of spinal cord shock, to help reduce the development of spasms. The acupuncture treatments were also helpful in the treatment of decubital ulcers. Additional studies have been published which support these findings. Honjo et al., (2000) from Japan, observed that needle stimulation of Bladder 33 (third posterior sacral foramina) significantly improved bladder incontinence. Cheng et al., (1998) from Taiwan, observed that cases who received electroacupuncture (acupuncture points CV 3, 4 and Bladder 32) achieved balanced voiding in fewer days than cases who received no acupuncture. Cases treated with acupuncture starting within 3 weeks after injury required significantly fewer days of treatments, than those treated with acupuncture after 3 weeks. Cases with complete spinal cord injury, either with pronounced detrusor-sphincter dyssynergia in upper motor neuron lesion or with persistent areflexic bladder in lower motor neuron lesion, were not affected by acupuncture. Yu (1993) from Beijing Medical University observed that 100 Hz electroacupuncture (EA) (2 times/day, 30 min/time) for 3 months had an antispastic effect on the limbs which was stable, but required additional long-term, follow-up treatments. He concluded that 100 Hz EA "decreased the excitability of the motor neurons in the anterior horns through the kappa opiate receptors, thus ameliorating the muscle spasticity of spinal origin." Low-level laser therapy (LLLT) may be used on acupuncture points to help reduce spasticity (Naeser & Wei, 1994, pp. 40, 41). The term LLLT refers to low-level lasers (5-500 mW, red-beam or near infrared, 600-1000 nm wavelength) which when applied to the skin produce no sensation and do not burn the skin. They have been observed to increase cellular adenosine tri-phosphate (ATP). LLLT has been used in studies to treat pain for over two decades (reviewed in Tuner & Hode, 1999). LLLT has also been used to reduce paralysis in stroke patients (Naeser et al., 1995). Laser acupuncture (and microamps TENS) can be applied to the hands
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
and feet, in a home treatment program to help reduce muscle spasms (Naeser & Wei, 1994, pp. 40, 41). They may also be used to treat decubitus ulcers (p. 59). References Cheng PT, Wong MK, Chang PL. A therapeutic trial of acupuncture in neurogenic bladder of spinal cord injured patients--a preliminary report. Spinal Cord 1998 Jul;36(7):476-80. Gao XP. Acupuncture for traumatic paraplegia. International Journal of Chinese Medicine 1984;1(2):43-47. Gao XP, Gao CM, Gao JC, Han CG, Han F, Han B, Han L. Acupuncture treatment of complete traumatic paraplegia Analysis of 261 cases. J. of Traditional Chinese Medicine 1996;16(2):134-137. Honjo H, Naya Y, Ukimura O, Kojima J, Miki T. Acupuncture on clinical symptoms and urodynamic measurements in spinal-cord injured patients with detrusor hyperreflexia. Urologia Internationalis 65 (4):190-195. Naeser MA, Wei XB. Laser Acupuncture - An Introductory Textbook for Treatment of Pain, Paralysis, Spasticity and Other Disorders. Boston, Boston Chinese Medicine, 1994, p.40. Naeser MA, Alexander MP, Stiassny-Eder D, Galler V, Hobbs J, Bachman D, Lannin L: Laser Acupuncture in the Treatment of Paralysis in Stroke Patients: A CT Scan Lesion Site Study. American Journal of Acupuncture, 23(1):13-28, 1995. Tuner J, Hode L. 1999. Low Level Laser Therapy: Clinical Practice and Scientific Background. Edsbruk, Sweden: Prima Books. Wang HJ. A survey of the treatment of traumatic paraplegia by traditional Chinese medicine. Journal of Chinese Medicine 1992;12(4):296-303. Yu Y. Transcutaneous electric stimulation at acupoints in the treatment of spinal spasticity: effects and mechanism. Zhonghua Yi Xue Za Zhi 1993 Oct; 73(10):594-5, 637.
Some Websites for Additional Information on Laser Acupuncture 1. Specializing in SCI: Pat Worth LAc, 21 Iron Gate Park Rd, Centerville, OH 937-439-9165 2. General Laser Acupuncture Information. A short website page written by M. Naeser, Ph.D., Lic.Ac., to help answer questions from licensed acupuncturists interested in laser acupuncture: http://gancao.net/ht/laser.shtml 3. Naeser Laser, Acupuncture Website for Carpal Tunnel Syndrome and Repetitive Strain Injury with color photos of how the therapy is performed. Written by M. Naeser, Ph.D., Lic.Ac., to help answer questions regarding laser acupuncture research to treat CTS and RSI: http://gancao.net/ht/cts.shtml 4. General Low-Level Laser Therapy Information (from Sweden and European sources). Extensive references and excellent information on low-level laser therapy research from around the world, written by the Swedish Laser Medical Society: www.laser.nu 5. Website with Low-Level Laser Therapy on Acupuncture Points, for Spinal Cord Injury. Written by Dr. Albert Bohbot, from France. Contains case histories, and videotapes of before and after treatments: www.laserponcture.net 6. Dr. Anu Makela, from Finland, who conducts low-level laser acupuncture research in many areas, including diabetes mellitus and neurological disorders:
[email protected] Peter Courtnage, Ph.D., Lic.Ac., Anchorage, AK, has studied with her in Finland:
[email protected]
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
7. Magnet Cap, developed by Agatha Colbert, M.D., Eugene, OR. Used to help reduce leg spasticity (or organ/stomach spasticity) in SCI patients (anecdotal experience, M.Naeser with SCI patients). May be ordered from Jayne Ronsicki, Lic.Ac., Hudson, MA
[email protected]
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
Summary Table. Acupuncture to Help Treat Paralysis in Spinal Cord Injury From M. Naeser report: “Neurological Rehabilitation: Acupuncture and Laser Acupuncture to Treat Paralysis in Stroke and Other Paralytic Conditions and Pain in Carpal Tunnel Syndrome," NIH Office of Alternative Medicine and the Office of Medical Applications of Research; Proceedings published by NIH in 1997 (pp. 93-109).
Authors
No. Cases Real Acupuncture
15/17 Cases, 88%
Yuci City Institute of Paralysis, Shanxi Province, China
17 Inpatients, with Complete Traumatic Paraplegia, Acute Cases, 1 Mo. postonset and Chronic Cases, 5 Yrs. postonset, Tx.’d over a 2 - 3 Yr. Period 82 Cases, Treated with Acupuncture/ ElectroAcptr., along the Bladder Meridian (paravertebral) for 5 Months
76/82 Cases, 93%, “Effective”
261 Cases, Treated beginning at 1 Mo. postonset to over 5 Yrs. postonset
249/261, 95%, “Effective”
Gao, 1984
Wang, 1992
Institute Health Preserv. Beijing, China
Gao, Gao, Gao, Han, Han, Han, Han, 1996
Number Control Cases Sham or No Acptr.
Yuci City Paralysis Institute, Shanxi Province, China
Number of Cases with Outcome Level of Beneficial Progress
Includes improvement in the following: Reduction in muscle spasms Increased level of sensation Improved bladder and bowel function Recommends beginning acupuncture as soon as possible after spinal cord injury, even during early stage of spinal cord shock, in order to reduce occurrence of spasms. Younger patients had better outcome.
Includes improvement in the following: Improvement in lower limb paralysis Improved bladder and bowel function
“Effective” defined as: Basic Recovery of functions of the nervous system with ability to walk freely, and almost voluntary urination (3% of cases). Marked Effectiveness with partial recovery of functions of nervous system, with ability to walk on crutches and restortion of urinary bladder reflex (35.2%). Improvement of functions of nervous system with some limb movement, defecation and/or urination (57.1%). Recommend beginning acupuncture as soon as possible after the spinal cord injury. Overall, Beneficial Progress Post-Acupuncture,
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
340/360 Cases = 94.4%
Jagan Nathan Vamanan PhD MSAR MAHMA PGA Acupuncture (Harvard) 18D Second Main Road, Netaji Colony West Velacherry, Chennai 600042 India +91 44 6527 1655/ 4355 9905
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[email protected]
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MD Acupuncture – doctoral dissertation march 2010 copyright material
MECHANISMS OF LOW LEVEL LIGHT THERAPY in ACUPUNCTURE - Laser Acupuncture Laser Acupuncture in Treatment of Paralysis in Stroke and Treatment of Carpal Tunnel Syndrome, Sports Injury, Post-op Pain, Wound Healing, Alopecia Areata ©
Table 4. Acupuncture/Laser Acupuncture for Peripheral Facial Paralysis (Bell’s Palsy) Duration No. Cases Duration of Real of Acupnctr Number of Cases with Outcome Level of Authors Acupuncture Paralysis Treatmnt Cured or Markedly Improved Gao & Chen, 1991
60 Cases
3 Days to 30 Years
Mild, n = 30 Severe, n = 30
10 Tx.’s, every other day
< 2 Mo., n = 40 > 2 Mo., n = 20
< 2 Mo. Duration: Cured, 92.5%; Excellent, 5%; Improved, 2.5%; > 2 Mo. Duration: Cured, 60%; Excellent, 20%; Improved, 15%; Failed, 5%
Beijing College of Traditional Chinese Medicine, Beijing, China Cui, 1992
Recommend starting Acupuncture soon postonset 100 Cases 9 were Recurrent Cases
Tangshan Hospital of Traditional Chinese Medicine, Hebei Prvince, China Liu, 1995
718 Cases
Shandong College of Traditional Chinese Medicine, Jinan, China Cheng, Zhao, Zhang, Yao, 1991 Chinese Academy of Traditional Chinese Medicine, Beijing, China
Wu, 1990
Overall, 59/60, 98% Mild Cases: Cured, 93%; Excellent, 7% Severe Cases: Cured, 70%; Excellent, 13%; Improved, 13%; Failed, 3%.
31 Cases 3 Mild 6 Moderate 22 Severe with Spasm eyelids, cheeks, both mouth corners
100 Cases
Puyang City People’s Hospital, Henan Province, China
5 to 40 Tx.’s, Daily
1 - 5 Days, n = 62 6-30 Days, n=3 1 - 6 Mo., n=6 > 6 Mo., n=2
90/100 Cases, 90%, Cured or Markedly Improved
94/100 Cases, Rec’d 30 Tx.’s over a 1-Month Period
All Cases, less than 4 Days
1-2 Months of Treatment
715/718 Cases, 99.6%, Cured or Marked Effect
1 Week to 20 Years
Acptr. plus Laser Acptr Red-Beam 15 mW, HeNe Laser 20 Min., Spasmodic Area
26/31 Cases, 84%, Basically Controlled, Markedly Effective or Improved.
27/31 Cases, >1 Year
