Biochemical and Biophysical Effects of Low Level Laser Irradiation MAL 2000, Helsinki, Finland Levon V. Gasparyan Currently the low level laser irradiation (LLLI) is widely applied in different branches of medicine. Several hypotheses are proposed to explain the mechanisms of low level laser therapy (LLLT). But unfortunately the mechanisms of positive influence of LLLI on the affected organism are not still completely understood. Various local and systemic medico-biological processes are take place at the ill organism under the influence of LLLI. The action of laser light of optimal parameters has normalizing influence bringing to the total rising of adaptive potential of the organism and to the acceleration of treatments. Initial phase of any biological and clinical effect of LLLI is the absorption of a quantum of energy by biotissues with induction of primary photophysical and photochemical processes. A process of absorption is depended for both from optical properties of biological object, and from parameters of laser irradiation. Ultraviolet, visual and infrared spectra radiations have biological activity. Photophysical processes lies in a basis of the photochemical reactions under the light influence. The potochemical reactions later cause the photobiological actions developing in an organism. The photophysical reactions are mainly determined by warming of the object to different degree (within the limits of 0.1-0.3°Ñ) and diffusion of heat in biotissues. The photochemical reactions are stipulated by exaltation of electrons in atoms of substance absorbing laser light. On molecular level it is expressed by photoionization of matter, its reduction or photooxidation, photodissociation of molecules, and in their rearrangement - photoisomeric change. The next step of interaction between laser light and organism is the development of the whole body positive reactions. Such reactions are used in LLLT for treatment. It was shown that the clinical effects of LLLI are connected with two maim processes. First of all, with a resonance absorption of light quantums by one or another molecules of chromophores (primary "acceptors"), and secondly with non-resonant thermal absorption of energy by biological molecules and water. The non-resonant absorption of energy caused local heating of macromolecules and membranous structures resulting in conformational changes and accordingly to modification of their function. The temperature difference is more expressed in biological membranes, that to lead the outflow of Na+ and K+ ions from the cells, opening of protein canals and increase of the transport of molecules and ions. The laser radiation causes heterogeneity of a temperature profile in biological tissues in consequence of irregular distribution of absorptive centers (biological membranes, proteins, ions in solution etc.) in tissues and cells. Such irregularity can substantially influence to metabolic processes in tissues and cells, since results in deformation of cellular membranes, and also in change of their electropotentials. The efficacy of LLLI influence on biotissues can be evaluated at microdeformations value of cellular membranes or change of membrane potentials. These values are determined by gradient of temperature, which one, in turn, depend on the difference of absorption coefficients for laser radiation in near-membrane areas. This implies, such parameters of radiation as polarization and coherency do not influence on efficacy and final effect of irradiation. The essential parameter appears to be a wavelength of radiation. Last is connected with an unequal spectral course of absorption coefficients of different substances, which are heterogeneously distributed in biotissues. In such treating the main advantages of laser light sources above all remaining become understandable: high spectral density and monochromaticity, which under condition of difference of absorption coefficients of different microstructures in biotissue ensure the high contrast of heterogeneity of a temperature profile. The dependence of biostimulation character on monochromaticity and non-importance of coherency and polarization of radiation was noted by several explorers. M. A. Kaplan (1997) has notified about the marked biological influence of lasers of different wavelengths. The stimulative effect is marked for irradiation of red light (630 and 640 nm). Under influence of blue (470, 480 and 490 nm) and yellow - orange (570 and 580 nm) light spectra the suppression of function activity of irradiated biological objects is take place. Other explorers find biological activity of radiation of the helium - cadmium laser with wavelength 441 nm, nitrogen laser with wavelength 337 nm. Biostimulative activity of green (500 nm) and violet (415 nm) light are also displayed. Enriching of blood microcirculation is detected under action of the gallium arsenide semiconductor laser (890 nm). The intensifying of biostimulative effects is revealed under combined influence of LLLI with the different spectral characteristics, in particular, under irradiation of red and blue laser light. N. F. Gamaleya supposed that the biological effects of LLLI are connected with natural processes of light regulation observed for animal. The mechanisms of similar processes are better studied on plants, for which not only facts of light-regulation, but also chemical nature of one of primary acceptors of light - phytochromes are established. On the principle about existence of animal photo-regulatory system, similar to phytochrome system of plants, it was suggested that biostimulative activity of radiation of the helium-neon laser is a consequence of coincidence of its spectral characteristics with bands of absorption of components of this system. The spectral dependence of bioeffect of different tested biological objects is congruent with the usual absorption spectrum of porphyrin compounds being a constituent of many relevant biochemical components of an animal organism - hemoglobin, cytochromes, series of enzymes (catalase, superoxide dismutase) etc. S. M. Zubkova (1987), G. E. Brill (1992) considered, that molecules of enzymes of antioxidant protection system can appear as primary acceptors of energy of HeNe laser irradiation. They thought about enzymes catalase which contains ferroprotoporphirin coferment complex, and superoxide dismutase (SOD) which contains a metal with variable valency (Cu2+, Zn2+ or Mn3+), strongly bound with protein. Both enzymes have absorption bands near 633 nm. The enhancement of activity of a catalase and SOD as a result of an irradiation by red light positively influences on antioxidant system of organism, results in decreasing the level of lipids peroxide oxidation with the subsequent physiological effects at numerous diseases. T. I. Karu (1989, 1994) proposed a hypothesis about interaction of LLLI with the respiratory chain components. She considered that, chromophores of HeNe laser light could be cytochromes a and à3 and cytochrome oxidase. The mechanism of activity of LLLI within the framework of this hypothesis includes such sequence of events: 1) At hypoxia under oxygen deficiency conditions the reduction of enzymes - respiratory chain transmitters and decrease of transmembrane potential of mitochondria are take place. 2) LLLI results in reactivation of these enzymes (for example, cytochrome oxidase), that restores a flow of electrons in the respiratory chain and shapes transmembrane potential of mitochondria. So increase of transmembrane potential in mitochondria, augmentation of production ATP in cells, activation of transport Ñà2+ are take place. The augmentation of ATP production and Ñà2+ ion concentration in the cell result in stimulation of endocellular processes. G. I. Klebanov et al. (1998) have formulated a hypothesis about the photodynamic mechanism of LLLI activity. The main points of the hypothesis can be presented as follows: 1. Chromophores for laser irradiation in red spectrum are the endogenous porphyrins, which are well known as photosensitizers. The content of porphyrins in an organism increases at many diseases and pathologic conditions not only in cell membranes but also in the blood plasma. 2. The porphyrins, absorbing energy of low level laser light, provoke photosensitized free-radical reactions, including lipid peroxide oxidation of cell membranes, so the amount of free forms of oxygen is growth. It leads to an increase in ionic permeability, including of Ñà2+ ions, across the leukocyte membranes. The content of Ñà2+ ions in extracellular volume is 3-4 degrees higher, than inside cells. Therefore small modification results in passing in the Ñà2+ ions from the extracellular space. 3. The increasing of the Ñà2+ ions content in cytosol of leukocytes starts Ñà-dependent processes leads to the priming of leukocytes. The essence of the priming phenomenon is that the interaction of leukocytes with any stimulant at a very low concentration which does not lead to the immediate activation of the cells still ends with an increase in the functional potential of the leukocytes. A manifestation of the increase is that the response of the cells to the subsequent stimulation at a normal concentration of another stimulant increases several times after this interaction. 4. Rising of the level of functional activity of the cell results in increase of production of different biologically active molecules (nitric oxide, superoxide anion-radical, hypochlorite ion, etc.). Some of them have bactericidal effect, and are also capable to influence microcirculation of the blood. The inducible NO-synthase appears in leukocytes, that results in synthesis of NO in leukocytes. This molecule is the precursor of so-called endothelium derived relaxing factor (EDRF) - which results in vasodilation and improvement of microcirculation contributes to the acceleration of the stage of reperfusion, which is the basis for the most favorable clinical effects of laser therapy. The is also a suggestion, that because oxygen has the absorption band near 640 nm, it actively absorbs red light, turns into singlet state and initiates oxidative processes in tissues. Examinations on human erythrocytes prove, that near infrared spectrum LLLI (700-900 nm) influences the deformability of the erythrocyte membrane. The mechanism of influence concludes the origination of primary damages of cytoplasm membrane by singlet oxygen generated as a result of a photoexcitation of molecular oxygen, having absorption bands near 586, 640,762, 1060 nm. It is necessary to pay attention to that fact, that all the examined above candidates for the role of primary acceptors of LLLI are not specific regulators of cell functions and their activation under laser irradiation cannot explain the integrated response showing in coordinated changes of many parameters of metabolism and functions of both cell and whole body levels. The laser light has positive influence on living cells and organisms at different pathologies. Is it possible that the basically outer regarding biosystem physical factor appear as the treating or normalizing factor? It is rather difficult to imagine. It is more logical to suspect that the outer physical factor works through the inner regulatory system of cells and organism, optimizing their operation and function. Such inner regulatory system, according to G. E. Brill (1997, 1999) could be the system of guanilate cyclase - cyclic guanosin monophosphate - NO-synthase. It is an indivisible system. The activation of a NO-synthetase results in an activation guanilate cyclase and further accumulation of cyclic guanosin monophosphate (cGMP). Guanilate cyclase catalyzes a biosynthesis of cGMP. The porphyrin complex (heme) having an atom of Mn instead of Fe one is the prosthetic group of guanilate cyclase molecule. The presence of the porphyrin complex in the structure of guanilate cyclase makes possible its absorption of red light quantums. The indirect evidence of probable participation of guanilate cyclase - cGMP system in realization of LLLI bioeffects is detected under certain conditions similarity of final effects of LLLI influence and guanilate cyclase - cGMP system activity. The cGMP effects includes vasodilation; stimulation of mitotic activity of cells, promotes an acceleration of an adhesion of wounds; and also delay of thrombocytes aggregation. The similar effects are registered under HeNe laser action too. NO is created in cells with the participation of NO-synthase enzyme. The prosthetic group of that enzyme contains compounds, which are capable to absorb red light photons. Endogenous NO activates guanilate cyclase as a result of interaction with heme. G. E. Brill (1997) suggested that the work of such photosensitive system could be connected with changes of intracellular level of cGMP. The cGMP molecule is a universal trigger molecule participating in transmission of photosignals both in specialized retinal cells, and in cells, not having specific photoreceptors. It was shown that the preliminary introduction of cGMP in cells takes out boosting effects of red light. It is very important the direct measurements indicated the rising of the content of cGMP in thrombocytes under HeNe laser irradiation. Thus, the mechanisms of action of low level laser irradiation in visual and infrared spectra on cells, tissues an organism as a whole include the processes taking place on cellular and molecular levels. Summarizing data of the modern studies, LLLI causes activation of energy synthesis processes in pathologically changed tissues with insufficiency of metabolism, increase of activity of main enzymes, reduction of oxygen consumption by tissues with simultaneously rising of mitochondria activity, rising of intensity of glycolysis in tissues and other. The secondary effects are the complex of adaptive and compensatory responses appearing as a result of resulting realization of primary effects in tissues, organs and the organism as whole. Among these responses it is necessary to mark the following: 1) Activation of a metabolism of cells and rising of their function activity; 2) Stimulation reparation (restoration) processes; 3) Antiinflammatory activity; 4) Activating of blood microcirculation and rising the level of trophic supply of tissues; 5) Analgetic activity; 6) Immunostimulative activity; 7) Reflex influence on functional activity on different organs and systems; 8) Rising of oxygen blood level. The laser therapy is inherent features of the justified pathogenic therapy method. At its application it is relevant the count not only general state of the organism, specificity of pathological process, its clinical manifestation, stages and form of diseases, but also associated diseases, age and professional features of the patient. The application of a phototherapy is the most successful in functional and reversible stages of illness, though the new procedures are applied at the treatment of heavier conditions and pathological process, at the presence of expressed morphological changes.