АКТИВАЦИЯ МИГРАЦИИ СТВОЛОВЫХ КЛЕТОК IN VITRO ПОД ВОЗДЕЙСТВИЕМ НИЗКОИНТЕНСИВНОГО ЛАЗЕРНОГО ИЗЛУЧЕНИЯ
Гаспарян Л.В., Брилль Г.Е. В настоящее время низкоинтенсивное лазерное излучение (НИЛИ) широко применяется при лечении различных заболеваний и патологических состояний. В недавних исследованиях была показана высокая миграционная способность стволовых клеток костного мозга в область повреждения. С учетом роли стволовых клеток в процессах репарации и регенерации, нами была выдвинута гипотеза о возможном повышении функциональной активности стволовых клеток при воздействии НИЛИ. Целью настоящего исследования явилось исследование активности миграции стволовых клеток под воздействием цитокина SDF -1?, а также НИЛИ красного и инфракрасного диапазонов.
Стволовые клетки FDCP - mix линии содержались в среде ISCOVE'S с добавлением 20% лошадиной сыворотки, пенициллина и стрептомицина в присутствии 20 нг/мл интерлейкина 3. Среда сменялась каждые 5 дней. Миграция стволовых клеток тестировалась в системе Transwell (Corning , NY). Стволовые клетки промывались в физиологическом растворе, после чего стандартное количество (2 x 10000 клеток в 600 мкл среды) помещалось в отсек №1 системы Transwell. В ряде экспериментов стволовые клетки предварительно подвергались облучению светом красного или инфракрасного лазера. В отсеке №2, изолированном от отсека №1 фильтром, помещалось 600 мкл среды. В ряде экспериментов к среде добавлялось 150 нг/мл SDF-1a. Количество клеток на фильтре, разделяющем два отсека, подсчитывалось после 4 часов инкубации во влажной атмосфере при 37 о С и 5% СО 2 .
Была исследована интенсивность миграции стволовых клеток в 6-и группах. В контроле (группа 1) исследовалась спонтанная миграция стволовых клеток из отсека №1 в направлении отсека №2, не стимулированных SDF-1a или лазерным излучением. Во второй группе стволовые клетки предварительно подвергались 15 минутному облучению светом красного полупроводникового лазера (660 нм, 20 мВт), а в группе 3 соответственно инфракрасного лазера (958 нм, 36 мВт). В четвертой группе определялась активность направленной миграции стволовых клеток по градиенту концентрации цитокина SDF-1a из отсека №2. В группах 5 и 6 исследовалось совместное воздействие SDF-1a и лазерного света красного (группа 5) и инфракрасного диапазонов (группа 6).
В результате исследования было выявлено, что спонтанная миграция стволовых клеток в контроле (группа 1) составила 1496,5±409 клеток (100%). Предварительное облучение стволовых клеток светом красного (группа 2) и инфракрасного (группа 3) лазера увеличило миграцию до 1892±283 (126%) и 2255,5±510 клеток (151%) соответственно. Таким образом, лазерное излучение стимулирует подвижность стволовых клеток. Как и ожидалось, в группе 4 была выявлена направленная миграция стволовых клеток к хемоаттрактору SDF-1a: 3365,5±489 клеток (225%) осело на фильтре. Показано также, что совместное влияние лазерного излучения и SDF-1a превосходит эффекты каждого из факторов в отдельности. Так, предварительное облучение клеток светом красного лазера увеличило направленную миграцию по градиенту SDF-1a до 5813±1199 клеток (388%), а облучение инфракрасным лазерным светом довело миграцию до 6391,5±540 клеток (427%).
Результаты данного исследования подтверждают гипотезу о чувствительности стволовых клеток к лазерному излучению. Активация направленной миграцию стволовых клеток по градиенту SDF-1a после лазерного облучения открывает перспективы для более широкого применения лазерного излучения в трансплантологии и гематологии. Наверх
СОВРЕМЕННЫЕ НАУКОЕМКИЕ ТЕХНОЛОГИИ № 3 2006
Full text PDF 
АВТОКОЛЕБАТЕЛЬНЫЙ ПРОЦЕСС, МОДИФИЦИРУЮЩИЙ
ФУНКЦИЮ ТРОМБОЦИТОВ В ЦЕЛЬНОЙ КРОВИ IN VITRO
Брилль Г.Е., Будник И.А., Гаспарян Л.В.
В опытах на цитратной крови 50 доноров обнаружены периодические изменения адгезии тромбоцитов на полистирене при высокой (1800 с–1) скорости сдвига. Средние значения периода колебаний, полученные при обработке нативных кривых и по данным вейвлет-анализа, составили около 35 мин. Отчетливая периодичность и высокая амплитуда циклов, бездекрементный характер ослилляций при отсутствии внешнего по отношению к исследуемой системе водителя ритма позволяют предположить наличие нового явления – существование автоколебательного процесса в цельной крови in vitro . Наверх
ИЗЛУЧЕНИЕ ПОЛУПРОВОДНИКОВОГО ЛАЗЕРА СТИМУЛИРУЕТ АНГИОГЕНЕЗ
Брилль Г.Е., Гаспарян Л.В.
Позитивное влияние низкоинтенсивного лазерного излучения (НИЛИ) на заживление ран и репарацию тканевых повреждений хорошо известно. Одним из механизмов, лежащих в основе стимуляции восстановительных процессов в тканях при действии лазерного излучения, может явиться активация роста и новообразования кровеносных сосудов – ангиогенез. В данном исследовании мы изучили влияние красного света полупроводникового лазера на ангиогенез, используя кольцевые препараты аорты крысы. Лишенные фиброзной и жировой ткани срезы аорты трижды промывались теплым стерильным раствором, содержащим 1% глутамин, пенициллин, стрептомицин и нистатин, и подвергались 10-минутному лазерному облучению (660 нм, 20 мВт). Далее препараты помещались в коллагеновый гель и выдерживались в сменяемой среде в течение недели во влажной атмосфере при 37С и 8% СО2 . Затем препараты фиксировались в 4% формалине и окрашивались генциан-виолетом. В ряде экспериментов к кольцевым препаратам добавлялся ростовой фактор сосудистого эндотелия (VEGF) в дозе 25 нг/мл. Проведенные исследования показали, что под влиянием VEGF происходит заметная активация ангиогенеза: площадь новообразованных сосудов достигает 1,3±0,24 мм2 , максимальная длина составляет 0,93±0,11 мм. После лазерного облучения также наблюдается отчетливая стимуляция новообразования сосудов: их площадь к концу наблюдения становится равна 1,9±0,29 мм2 , максимальная длина - 0,75±0,10 мм (p>0,2 по сравнению с VEGF). Следовательно, НИЛИ оказывает стимулирующее влияние на ангиогенез и этот эффект по степени выраженности сопоставим с действием важнейшего медиатора ангиогенеза VEGF . Если лазерное облучение препарата предшествовало аппликации VEGF, то наблюдался наиболее выраженный стимулирующий эффект. При этом площадь новообразованных сосудов достигает 6,98±0,88 мм2 , максимальная длина - 1,7±0,23 мм, что превышает результат, наблюдаемый как при действии только ростового фактора, так и только лазерного облучения. Таким образом, лазерное излучение красной области спектра само по себе оказывает выраженное стимулирующее влияние на процессы ангиогенеза и кроме того потенцирует эффекты ростовых факторов, в частности, VEGF . Это свойство лазерного излучения объясняет эффективность лазеротерапии многих заболеваний, в основе которых лежат явления гипоксии и ишемические поражения тканей. Наверх
СРАВНИТЕЛЬНЫЙ АНАЛИЗ ВЛИЯНИЯ КРАСНОГО, ИНФРАКРАСНОГО И СИНЕГО СВЕТА НА АДГЕЗИЮ И АГРЕГАЦИЮ ТРОМБОЦИТОВ
Гаспарян Л.В., Брилль Г.Е.
Проведен сравнительный анализ эффектов излучений разных длин волн на адгезию и агрегацию тромбоцитов на полистирене при высокой скорости сдвига. 1 мл цитратной цельной крови облучался с помощью сменных головок светодиодного облучателя «Рубин-МЦ»: синий свет (470 нм), ИК (870 нм) и красное (630 нм) излучения. Облучение проводилось в течение 20 мин при мощности на выходе излучателя - 10 мВт и одинаковой плотности мощности. После окончания 20-минутного облучения образцы крови исследовались через 1, 10, 20 и 30 мин. В качестве контроля использовалась кровь того же донора, не подвергавшаяся фотовоздействию. 130 мкл исследуемой крови помещались в полистиреновую кювету, которая устанавливалась в прибор Cone and Plate(let) Analyzer. В течение 2 минут образец подвергался воздействию скорости сдвига (shear rate) = 1800 с-1 . После промывки проводилась окраска препарата в течение 2 мин красителем Май Грюнвальд. Препарат подсыхал на воздухе и анализировался с помощью имидж-анализатора. Рассчитывались 2 параметра: площадь, занимаемая адгезированными объектами и средний размер объектов.
Проведенные исследования показали, что облучение крови ИК-светом при выходной мощности 10 мВт в первые 10 минут вызывает достоверное увеличение адгезии и агрегации пластинок, однако к 20-й минуте и далее эти изменения нивелируются и поведение тромбоцитов не отличается от контроля. Изменения функции тромбоцитов, вызываемые синим светом, качественно весьма сходны с таковыми, вызываемыми ИК излучением. Здесь также наблюдается начальное (в течение первых 10 минут после фотовоздействия) повышение адгезии и агрегации тромбоцитов, особенно выраженное на 10-й минуте. Однако и при облучении синим светом эти изменения обратимы и к 20-й минуте происходит восстановление функции пластинок. Иная закономерность прослеживается при облучении крови красным светом. Обращает на себя внимание резкое и высокодостоверное (p<0,001) угнетение адгезии и агрегации пластинок, наблюдаемое на 1-й минуте после прекращения облучения. Эта ингибиция функции пластинок кратковременна и обратима: уже на 10-й минуте адгезия и агрегация тромбоцитов нормализуются и даже имеется некоторая тенденция к активации пластинок. Таким образом, сопоставление биоэффектов одинаковой дозы излучений разных длин волн, с одной стороны, позволяет выявить сходство этих эффектов (ИК и синий свет), а с другой стороны, свидетельствует о наличии особенностей реакции (по крайней мере для тромбоцитов), что характерно для влияния красного света. Наверх
ВЛИЯНИЕ СВЕТОДИОДНОГО ИЗЛУЧЕНИЯ НА ФУНКЦИОНАЛЬНУЮ АКТИВНОСТЬ ТРОМБОЦИТОВ
Гаспарян Л. В., Дементьева И. Н.
Появление светодиодных излучателей повышенной яркости позволило создать фототерапевтические аппараты, способные генерировать свет различных длин волн. Дерябин Е.И. (1997) установил, что светодиодное излучение красного диапазона позволяет достигать лечебных эффектов, адекватных эффектам красного лазерного воздействия, которое широко применяется в клинике.
Целью нашего исследования было сравнительное изучение влияния излучения светодиодных матриц с различными длинами волн на индуцированную агрегацию тромбоцитов в цельной крови крыс.
Материалы исследования. Опыты проводились на белых крысах-самцах линии Wistar. Исследования индуцированной агрегации тромбоцитов проводились на импедансном агрегометре АИ-300. В качестве индуктора агрегации применялись АДФ в концентрации 2,55?10(-4) моль/л и адреналин в концентрации 10(-4) моль/л. Пробы крови облучались светодиодными матрицами (фирма Kingbright , США), излучающими свет с длинами волн 430 нм, 565 нм, 595 нм, 660 нм и 880 нм. В качестве контроля использовалась цельная крысиная кровь без облучения.
Результаты исследования. Во всех опытах с применением света различных длин волн, но одинаковой дозы (0,045 Дж/см 2 ) наблюдалось уменьшение интенсивности агрегации, наиболее выраженное при облучении крови синим и красным светом - на 37% и 45% соответственно (p<0,05). Для выявления зависимости доза-эффект были проведены исследования в диапазоне доз от 0,015 до 2,48 Дж/см2 . При использовании светодиодной матрицы синего света при дозах облучения 0,13 Дж/см2 , 0,26 Дж/см2 и 0,39 Дж/см2 интенсивность адреналининдуцированной агрегации уменьшилась на 24%, 45% и 49%, а АДФ-индуцированной агрегации - на 10%, 14% и 27% соответственно. Аналогичная динамика - уменьшение интенсивности агрегации при увеличении дозы - отмечена также при применении светодиодной матрицы красного цвета. При облучении зеленым светом с дозами 0,03 Дж/см2 , 0,06 Дж/см2 , 0,09 Дж/см2 наиболее выраженное подавление интенсивности адреналининдуцированной и АДФ-индуцированной агрегации на 38% и 46% соответственно (p<0,05) отмечено при применении минимальной дозы. Аналогичные результаты отмечены при облучении крови светом желтого и инфракрасного диапазонов. Таким образом, влияние светодиодного излучения в диапазоне длин волн 430 - 880 нм на агрегацию тромбоцитов было однонаправленным, но выраженность его зависела от длины волны и дозы.
Обсуждение. Наши данные согласуются с литературными данными об ингибирующем эффекте излучения красного света на агрегационную функцию тромбоцитов. Брилль А.Г. (1998) объяснил данный эффект увеличением в облученных тромбоцитах уровня цГМФ за счет активации его синтеза при участии гуанилатциклазы. Нельзя исключить также первичное повышение уровня NO в цельной крови с последующим проявлением антиагрегантных свойств данного соединения. Наверх
ROLE OF L-ARGININE IN THE BIOLOGICAL EFFECTS OF BLUE LIGHT
Makela A.M.
Some of the therapeutic effects of light can be considered to be due to the changes in the metabolism of L-arginine in the cells.
Increased plasma levels of arginine lead to enhanced secretion of several hormones including insulin, glucagon, growth hormone, prolactin, and adrenal catecholamines.
Production of L-arginine is regulated by several factors. L-arginine is produced from citrulline in the kidney cells and ornithine in the liver cells. Activation of carbamoyl phosphate synthetase will also produce arginine from glutamate through the carbamoyl-phosphate pathway. Glutamate reacts strongly to 537 nm and 712 nm, 806 nm, 865 nm and 945 nm wavelengths.
NO is formed from L-arginine by oxidation of its guanidino-nitrogen terminal by NO synthase. NO synthase is primarily a cytosolic enzyme requiring calmodulin, Ca 2+ , a-nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH), and tetrahydrobiopterin, and has similarities with cytochrome P450 enzymes. These all have absorption maxima between 446 nm and 652 nm. Several isoforms of the enzyme occur in endothelial cells, as well as in platelets, macrophages, vascular smooth muscle cells, and the brain. L-arginine has also been shown to be the unique substrate for the production of agmatine. A gmatine has potential as a treatment of chronic pain, addictive states and brain injury. It also competitively inhibits the activity of all isoforms of nitric oxide synthase , protecting the body from excess effects of NO . This reaction can be regulated by cytochrome P450 and light at 450 nm, cytochrome a gamma with light at 436nm, and cytochrome a alpha with light at 630nm. Recent evidence suggests that agmatine, which is an intermediate in polyamine biosynthesis, might be an important neurotransmitter in mammals. Agmatine is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake, released by depolarization, and inactivated by agmatinase. Agmatine binds to alpha2-adrenoceptors and imidazoline binding sites, and blocks NMDA receptor channels and other ligand-gated cationic channels. Furthermore, agmatine inhibits nitric oxide synthase, and induces the release of some peptide hormones. As a result of its ability to inhibit both hyperalgesia and tolerance to, and withdrawal from, morphine, and its neuroprotective activity, agmatine has potential as a treatment of chronic pain, addictive states and brain injury and protects against the harmful effects on excess NO.
It is proposed that the regulation of L-arginine turnover by the use of light at blue wavelengths between 400 and 510 nm can be the explanation for the observed effects of blue light: lowering of blood pressure, pain killing effect, regulating insulin production, anti-inflammatory action, and possible effects on the release of stem cells. Наверх
THEORETICAL BACKGROUNDS FOR LIGHT APPLICATION IN DIABETES
Makela A.M.
Glucose can act as an oxidizing agent in glycation breakdown depending on the composition of surrounding molecules. Glucose reacts nonenzymatically with protein amino groups to initiate glycation, the early stage of the Maillard reaction, leading to crosslinking and browning of the proteins via the formation of advanced glycation end products (AGEs). The AGEs are responsible for various biochemical in tissues which can lead to the development of several complications in diabetes: neuropathy, angiopathy.
The monocyte macrophage plays an important role in this process both by removing the senescent molecules that have accumulated AGEs over time and by initiating the steps that lead to new protein synthesis and tissue remodelling. One of the most important features of the macrophage is its ability to produce and release NO and SOD. The irradiation of macrophages by red light result in a dose-dependent increase in NO production and SOD activity and, laser irradiation of cells in the red range activates the synthesis of SOD and inducible NO-synthase de novo due to photosensitized initiation of free radical reactions.
NO synthase is primarily a cytosolic enzyme which has similarities with cytochrome P450 enzymes. These all have absorption maxima between 446 nm and 452 nm. Several isoforms of the enzyme occur in endothelial cells, as well as in platelets, macrophages, vascular smooth muscle cells, and the brain.
The start of pathogenic levels of Islet cell antibodies (ICA-IgG) may precede the clinical onset of diabetes by several years, even in children. Several attempts have been made to influence the course of type I diabetes by immunotherapy. Plasmapheresis, prednisone, and interferon have proven unsuccessful or only partially successful. Successful methods of preventing diabetes in Worchester rats have been neonatal thymectomy, antiserum to lymphocytes, bone marrow transplantations, and cyclosporine. This demonstrates the strong immunological background of the disease process. Type I diabetic patients have been shown to have inhibition of migration of leucocytes specific for antigens of the endocrine pancreas. Phagocytic white blood cells employ the myeloperoxidase H202 system to generate reactive oxygen intermediates that kill invading bacteria, viruses, and tumour cells. Partially reduced oxygen species are also potentially damaging to cellular lipids, nucleic acids, and proteins; the production of such species by activated phagocytes has been implicated in the damage of normal tissues under pathological conditions. The initial pathway for oxidant generation involves a membrane associated NADPH oxidase that reduces oxygen to superoxide anion, which then dismutates to form H202 . Myeloperoxidase, a secreted heme protein, amplifies the toxic potential of H202 by producing reactive intermediates. Production of myeloperoxidase is inhibited by irradiation at 633 nm 660 nm, 820 nm, 880 nm and 950 nm, of which 660 nm appears to have the strongest effect.
By regulating the amounts of active macrophages, NO, SOD, Myeloproxidase, and the activity of cytochrome P450 and many other substrates by light, it is possible to regulate glucose and AGE breakdown and prevent development of complications of diabetes. Наверх
INFLUENCE OF LOW LEVEL LASER RADIATION ON MIGRATION OF STEM CELLS
Gasparyan L.V., Brill G.E., Makela A.M.
The long term effects of low level laser therapy can involve treatment mechanisms connected with activation of stem cells.
In the current study migration of stem cells was tested under the influence of laser light alone as well as in case of combined influence of light and stromal cell-derived factor-1a (SDF-1a). This cytokine plays a role in lymphocyte trafficking, hematopoietic progenitor cell and stem cell homing.
To investigate the light influence on stem cells, we analyzed factor-dependent cell-Patersen (FDCP)-mix multipotent progenitor cells.
Migration of the stem cell line was tested using Transwell system (Corning, NY) under influence of red diode laser (?=659.6 nm, 19.5 mW) or infrared diode laser (?=958 nm, 36 mW) during 15 min at continuous wave, as well as in case of applying 150 ng/ml SDF-1a.
Group 1 cells were a group of negative control, group 2 cells received only red light irradiation, while group 3 cells had IR light irradiation. Group 4 cells were treated with 150 ng/ml SDF-1a. Group 5 cells were irradiated with red laser light in addition to 150 ng/ml SDF-1a, and group 6 cells by IR light and 150 ng/ml SDF-1a.
The count of migrated cells was 1496,5±409 (100%) in case of negative control. Red and IR laser light increased migration activity of stem cells up to 1892±283 (126%) and 2255,5±510 (151%) accordingly. Influence of SDF-1a was more significant, than effects of light irradiation alone 3365,5±489 (225%). Combined effects of light irradiation and SDF-1a were significantly stronger 5813±1199 (388%) and for SDF-1a and red laser light, and 6391,5±540 (427%) for SDF-1a and IR laser light irradiation.
Preliminary study results proved, that laser light irradiation can activate stem cell migration in vitro. The results are more reliable in the case of combined application of light and SDF-1a . These results are giving ground to consider that stem cell reactions on light irradiation can be one of the factors of light therapy. Наверх
ACTIVATION OF ANGIOGENESIS UNDER INFLUENCE OF RED LOW LEVEL LASER RADIATION
Gasparyan L.V., Brill G.E., Makela A.M.
Activation of angiogenesis under laser irradiation is one of the feasible explanations for long lasting treatment effects of laser therapy. Vascular endothelial growth factor (VEGF) is one of the most important growth factors for endothelium. VEGF induces angiogenesis and endothelial cell proliferation and it plays an important role in regulating vasculogenesis.
The aim of the current study is the investigation of red low level laser light influence on angiogenesis study in vitro, and comparison with effects of VEGF application.
Thoracic aortal rings were prepared from Sabra rats. The rings were than washed 3 times in sterile warm Bio-MPM medium containing 1% glutamin, penicillin, streptomycin, and nystatin. Samples of group 1 served as control, group 2 samples were incubated with VEGF, group 3 samples were irradiated with low level laser (660 nm, 20 mW) in a drop (10 µl) of the medium during 10 min, and group 4 samples were incubated with VEGF and received 10 min of laser irradiation.
The plates were maintained for 1 week (37° C, 8% CO2 , humidified atmosphere) and the medium was changed every two days. After this time period, the cultures were fixed with 4% formalin during 24h and stained with 0.02% gencyan violet solution in ethanol ( Sigma , Israel ). The stained samples were photographed by the camera connected to the microscope.
Low level laser irradiation activates the process of angiogenesis. In the control group (without VEGF or laser irradiation) angiogenesis of new vessels was not detected. Low level laser irradiation promoted angiogenesis. The area covered by new vessels was 1,9±0,29 mm 2 and the maximal length of vessels was 0,75 ± 0,10 mm. No statistical difference was discovered between laser irradiation and VEGF application. After combined influence of VEGF and laser light irradiation the area covered by new vessels was 6,98 ± 0,88 mm 2 and the maximal length of vessels was 1,7 ± 0,23 mm. So, laser irradiation can intensify effects of VEGF on angiogenesis. Наверх
INVOLVEMENT OF CYTOCHROME P450 IN MODULATION OF
LIGHT-INDUCED REACTIONS IN MAMMALIAN CELLS
Anu Makela
For years, scientists have been researching the effects of red light on human cells. However, recent studies show that different wavelengths can have various effects on cells not necessarily always seen with red light wavelengths. One apparently central factor in these reactions is cytochrome P450 and its relation to the production or activation of oxygen, SOD, NO and H2O2 .
In mammalian cells, cytochromes P450 serve as terminal electron acceptors in electron transport systems. The cytochrome P450 proteins contain a single iron protoporphyrin IX prosthetic group, which binds oxygen. One of the forms of regulating the functioning of Cytochrome P450 is the use of light of different wavelengths.
Cytochrome P450 monooxygenase systems can act as an intermediate in the activation of oxygen in hydroxylation reactions in which the function of superoxide dismutase is to catalyze the dismutation of superoxide to oxygen and water.
It has been demonstrated that SOD specifically catalyses the dismutation of superoxide and that its radical, if its concentration is elevated, may react with hydrogen peroxide to form a much more reactive hydroxyl radical.
SOD levels have been found to correlate with such diseases as ALS, MS, reactive arthritis, autoimmune diseases and prostate and breast cancer.
The function of SOD can be regulated by activating the redox sites of copper and zinc by their absorption maxima at 404 nm, 450 nm, 584 nm, 620 nm, 632 nm, 680 nm, 685 nm, 760 nm, 780 nm or 820 nm. It would seem that the possibility of controlling the advance of diseases connected with SOD level changes would be possible by the use of light. Наверх
BIOCHEMICAL EFFECTS OF LIGHT IRRADIATION AND KININS
Anu Makela
The primary changes in cells induced by light are followed by biochemical reactions which continue in darkness. These reactions are associated with the changes of cellular homeostasis, specifically the cellular redox state. The change of tissue pH is one of the regulating factors for kinin production.
Kinins are potent vasodilator peptides generated in blood and tissues. Naturally occurring kinins and their active metabolites exert various biologic actions. Kinins are among the most potent activators of the arachidonic acid cascade, and they promote the release of prostaglandins and prostacyclins. Kinins also stimulate the release of histamine and 5-hydroxytryptamine from mast cells. They modulate the motility and the function of leukocytes, macrophages, fibroblasts, and other cells. Kinins are also implicated in the pathogenesis of inflammation, tissue reactions to injury, and tissue repair. Kinins release a potent endogenous vasodilator from the endothelium that reduces the arterial smooth muscle tone, increasing the blood flow to organs and reducing the systemic blood pressure. Kinins increase capillary permeability, by diminishing the size of the endothelial cells; they stimulate the veins; and also modulate the functions of blood and tissue cells.
Therefore, light regulation of kinin production can explain some of the photobiological effects of light irradiation. Наверх
MODIFICATION IN RELATIONSHIPS IN THE SYSTEM ENDOTHELIAL CELL – BLOOD PLATELET
BY LOW LEVEL LASER IRRADIATION
G.E.Brill , L.V.Gasparyan, A.M.Makela
Nowadays laser irradiation is widely used in different areas of medicine. Blood cells, chemical components of plasma, cells of vascular walls are subjects of light exposure during the intravenous laser blood irradiation treatment.
In the current study the influence of the laser light on endothelial cells and platelets was investigate. HUVEC endothelial cell culture was irradiated by HeNe laser. The expression of E-selectin was detected as a sign of activation of adhesive function of the cells after 20 min of laser irradiation. Higher adhesion rate of intact platelets to irradiated endothelial cells was revealed. So, activation of endothelial cells and higher ability of adhesion of platelets were results of laser irradiation of endothelial cells.
Platelet response to laser irradiation was completely different. Inhibition of platelet activation by TRAP due to decrease of expression of P-selectin and weaker ability of binding fibrinogen by GPIIbIII membrane receptor were detected. Inhibition of aggregation induced by different inductors (ADP, ristocetin, adrenalin, collagen) and inhibition of adhesion on extracellular matrix, collagen covered surfaces as well as intact vascular endothelium was detected. The inhibitory effect of laser irradiation on platelets was mediated by photoactivation of guanilatcyclase, higher amount of cGMP and inhibition of intracellular reactions with proteincinase C participation.
As a conclusion, the different way of reacting of endothelial cells and platelets on laser irradiation is an important factor, supporting circulatory hemostasis during intravenous laser blood irradiation. Наверх
THE INFLUENCE OF LED IRRADIATION AT DIFFERENT WAVELENGTHS
WITH ANTIOXIDANTS ON FUNCTIONAL ACTIVITY OF BLOOD PLATELETS
Gasparyan L.V., Dementjeva I.N., Makela A.M.
The aim of the study was to compare the effects of LED light irradiation at different wavelengths on induced blood platelet aggregation and to determine the influence of light irradiation in the presence of different antioxidants.
LED clusters ( Kingbright , USA ) of different wavelengths were used to irradiate Wistar rat blood. Platelet aggregation was measured by impedance aggregometer. ADP and adrenalin were used as aggregating agents.
In the first series of experiments blood samples of test groups were exposed to LED irradiation (430, 565, 595, 660 and 880 nm) prior to aggregation induction. Control group blood was tested without light irradiation. In all test groups inhibition of aggregation was recorded. The strongest inhibition of aggregation in case of a single dose of irradiation (0,045 J/cm2) was detected after blue and red light exposure (37% and 45% accordingly, p<0,05). It was also noted, that in case of red and blue light, inhibition of aggregation got stronger with the increase of irradiation dose, while in case of green, yellow and IR light the weakest dose caused the strongest inhibition.
In the second series of experiments the blood samples of test groups were (a) exposed to red LED irradiation (660 nm), (b) treated with antioxidants, (c) treated with antioxidants and exposed to red LED irradiation (660 nm) prior to aggregation induction. Catalase by itself increased the level of aggregation (p<0,001), whereas it decreased significantly in case of combined action of catalase and red light.
Presented data correlate with the results of Brill G.E. (1998) findings showing inhibitory effect of red light irradiation on platelet aggregation as a result of increase of cGMP amount in platelets. It is also possible that the primary effect of the light irradiation is the increase of the level of NO in the blood with secondary inhibition of blood platelet activity. Results clearly demonstrated dose dependence according to individual wavelengths. Наверх
10th Congress of the European Society for Photobiology, Vienna, Austria, 2003
THE INFLUENCE OF LED IRRADIATION AT DIFFERENT WAVELENGTHS ON FUNCTIONAL ACTIVITY OF BLOOD PLATELETS
Gasparyan L.V., Dementjeva I.N, Makela A.M.
The aim of the study is to compare the effects of LED light irradiation of different wavelengths on induced blood platelet aggregation. LED clusters were used as sources of light. The study was conducted on Wistar rats. The level of induced blood platelet aggregation was measured by impedance aggregometer. ADP and adrenalin were used as aggregating agents. The blood samples of test groups were exposed to LED irradiation (430, 565, 595, 660 and 880 nm) prior to aggregation induction. The control group blood was tested without light irradiation. In all test groups inhibition of aggregation was recorded.
The strongest inhibition of aggregation in case of a single dose of irradiation (0,045 J/cm2) was detected after blue and red light exposure (37% and 45% accordingly, p<0,05). It was also noted, that in case of red and blue light, inhibition of aggregation became stronger according to increase of irradiation dose, while in case of green, yellow and IR light the weakest dose caused the strongest inhibition.
Presented data conforms with the results of Brill G.E. (1998) showing inhibition effect of red laser light irradiation on blood platelets aggregation due to activation of guanilatecyclase and increase of cGMP amount in irradiated platelets. Наверх
WALT 2-nd Congress (Kansas City, USA), 1998
LOW LEVEL LASER THERAPY OF MALE GENITAL TRACT CHRONIC INFLAMMATIONS
Levon V. Gasparyan, Samvel R. Grigoryan
INTRODUCTION
The authors summarized their experience of treating patients with mail genital tract chronic inflammations caused by sexually transmitted diseases (STD) (Chlamidial infection, Trichomoniasis, etc.) using low level laser therapy (LLLT). Evolution of antibiotic resistant strains, spreading of chronic latent infections complicated the treatment and required several courses of medicament therapy.
LLLT helps to overcome the problems. LLLT of such patients includes laser irradiation of blood, prostate gland and skin projections of male genitals. Laser irradiation activates immune system, increases the permeability of prostate gland tissues for antibiotics, has antiinflammatory, analgetic properties, activates spermatogenesis, activates local microcirculation, and sex function.
The obtained results proved that the combination of LLLT with medicament therapy provides better and faster treatment results.
REVIEW AND THEORY
The incidence of chronic and latent forms of STDs has risen recently despite advances in diagnosis and treatment. Changes in sexual behavior, imperfect and late diagnosis, lack of adequate laboratory facilities, poor cultural examinations, incomplete treatment as a result of usage of low quality and adulterated antibiotics, cases of non-professional and self-treatment, reinfection from infected, but not treated sex partners, evolution and spreading of antibiotic resistant strains complicated the treatment of STDs.
Several groups mentioned beneficial effects of LLLT in the treatment of male genital tract diseases. Sato et al. (1984) demonstrated that laser irradiation had potential to increase sperm motility in vitro. Therapeutic laser applied directly to testes at a dose of 1.3 J/cm2 has been successfully used in treatment of infertility (Hasan et al. 1989). Using HeNe laser applied directly to the scrotum, Miroshnikov and Reznikov (1989) found an immediate reduction in the pain, swelling, pyrexia and other symptoms of acute epididimitis. LLLT also reduced the necessity for surgical interventions. This group also reported about positive effects of HeNe laser irradiation in treatment of chronic intractable urethritis. LLLT produced reduction in pain, improvement in microscopic investigations, especially as regards the number of leukocytes. Such studies reported no side effects or complications so dangers associated with laser irradiation of or near the gonads are minimal. Koultchavenia (1997) found higher concentration of antibiotics in kidneys and prostate gland after local laser irradiation. Some studies showed the increase of sensitivity of microorganisms towards antibiotics after laser irradiation (Gorochov 1991, Avdoshin 1992). Kartachov (1994) mentioned that when some patients with bacterial latent urethritis started LLLT, Trichomonas vaginalis was found out in cultural studies. So laser irradiation could act like provocative test and help to diagnose latent STDs more precisely.
Duplik (1993) introduced a parameter "Specific Power Density" (mW/cm3) connected with both power characteristics of laser and optical properties of irradiated blood. Ovsjannikov (1997) suggested that calculation of energy absorbed in joules per cubic cm (J/cm3) of target organ tissues was physically more correct, than calculation of energy density in J/cm2 of irradiated skin.
The authors aimed to study the role and parametres of LLLT in the complex treatment of patients with male genital tract chronic intractable inflammations, caused by some STDs.
PROCEDURES
78 patients aged from 21 to 54 suffering from mail genital tract chronic inflammations (urethritis, prostatitis, vesiculitis, epididymo-orchitis) were divided to a medicament therapy group (group I, 36 patients) and medicament plus laser therapy group (group II, 42 patients). Subjective and objective methods of investigation were used to diagnose diseases and evaluate the treatment. Laboratory studies included microscopic and cultural examinations of specimens of urethral discharge, urine, prostatic fluid, sperm, blood, as well as ultrasound and other examinations. Provocative tests are used to make latent STDs diagnosis more reliable. Posttreatment cultures were studied to establish that the treatment was adequate.
Each patient of group II received 10-12 procedures of laser irradiation, including 3-4 LBI and 7-8 procedures of laser irradiation of skin projections of mail genitals and inguinal areas as well as transrectal irradiation of prostate gland. HeNe laser (632.8nm/ 2mW at the end of light-guide, inserted into a vein) was used for intravenous LBI. Pulsed IR laser cluster probe (12x890nm/5W pulse power) was used for irradiation of skin projections of male genitals. Pulsed IR diode laser probe (890nm/15W pulse power) with transrectally inserting fiberoptic probe was used for prostate gland irradiation. LLLT started simultaneously with antibiotic, antiinfalammatory and immune therapy.
RESULTS
Patients of group II reported (statistically reliable) faster resolution of symptoms of diseases (pain, dysuria, sex disorders), had better and more stable laboratory investigation reports (reduction and normalisation of leukocytes, elimination of microorganisms from specimens of urethral discharge, prostatic fluid, urine, sperm), grew of quantity and motility, as well as in percentage of normal forms of spermatozoa. Lower relapse rate for patients of group II (11%) than for patients of group I (18%) was recorded. No side effects or complications were detected. The authors estimated therapeutic 3D energy density for transrectal and cluster probe prostate gland irradiation.
The combination of intravenous LBI with local skin and prostate gland irradiation proved to be the most effective method of LLLT. The combination of medicament therapy and LLLT provides better and faster resolution of the symptoms and normalization in results of instrumental examinations.
DISCUSSION
Obtained results proved that laser therapy is an acceptable tool in the complex treatment of male genital tract chronic inflammations, caused by STDs. Our studies supported the opinion that acceleration of treatment was mainly the result of activation of immune system and increase of concentration of antibiotics in target organs.
More works are required to study laser light distribution in different tissues to determine the most effective 3D energy density for genital tract organs irradiation, combination of different wavelength lasers and methods of irradiation. Additional investigations are required to determine parametres of laser irradiation as the most promoting activity of immune system, increasing both microorganism sensitivity towards antibiotics and prostate gland permeability for antibiotics to create higher concentration of drugs in genital tract organs as well as to combine UV laser bactericidal action with the effect of antibiotics to achieve maximal medical effect. Наверх
WALT 2-nd Congress (Kansas City, USA), 1998
INVESTIGATION OF SENSATIONS, ASSOCIATED WITH LASER BLOOD IRRADIATION
Levon V. Gasparyan
INTRODUCTION
The author summarized his experience of treating patients with some angiological, surgical and urological disorders and concentrated on study of sensations, caused by low level laser blood irradiation (LBI). It was shown, that sensations in damaged areas during LBI procedure had reliable character for patients, suffering from peripheral atherosclerotic disease, trophic ulcers and septic wounds. It was supposed that sensation of warmth was the result of activation of local blood microcirculation. Such patients showed better treatment results. No sensations caused by LBI were revealed for patients with chronic prostatitis.
The author compared therapeutic effects of different lasers. It was shown that intravenous (632,8 nm) and transcutaneus LBI (850 and 890 nm) in certain dosage caused identical sensations.
Obtained results help to determine more accurately individually required dosage of LBI, to use noninvasive transcutaneus LBI instead of invasive intravenous one, to improve and accelerate the treatment.
REVIEW AND THEORY
Low level LBI is one of the most perspective methods of low level laser therapy. Studies proved several bands of laser irradiation stimulated DNA, RNA and protein synthesis. Some scientists supposed that these wavelengths activated special photoreceptor system, similar to photo-acceptor system of plants (Karu 1997).
LBI is used in the treatment of several disorders more than 20 years (Korochkin et al. 1995). The positive influence of LBI was proved, using special instrumental methods of investigations. LBI had several positive effects, such as antiinflammatory effect, activation of immune system, protection of vessels, improvement of blood microcirculation and tissue trophic processes, activation of regeneration and reparation (Sirenko et al. 1992). Better state of vascular wall, atherosclerotic plaque size reduction, improvement in blood microcirculation were observed among patients with atherosclerotic vascular disease after transcutaneus infrared (IR) LBI (Kaplan et al. 1997). Comparison between various kinds of lasers in the treatment of stenocardia proved that the best effect was obtained when IR transcutaneus or red intravenous LBI were applied (Kostin 1997).
LBI has two main versions: intravenous and transcutaneus. Intravenous LBI with HeNe lasers (632.8 nm) is the mostly used method. The laser irradiation is delivered via a thin light-guide, introduced with a needle into a vein. IR lasers (820-860, 890, 1200-1300 nm) are used for transcutaneus LBI. IR radiation penetrates tissues deeper, so direct contact between light-guide and blood is not required. Laser light for transcutaneus LBI is delivered to a skin projection of cubital vein or radial artery (Gelfant 1992, Duplik 1995).
Several patients reported feelings of warmth and tingling in those areas of skin where laser irradiation was applied. But no paper concerning sensations caused by LBI was found. The author aimed to study sensations associated with LBI to find out interconnections between sensations and clinical results of treatment, to reveal sensations typical for different diseases, to find out similarities and differences of influence between various lasers on organisms.
PROCEDURES
89 patients with angiological, surgical and urological disorders (peripheral atherosclerotic disease, trophic ulcers, septic wounds, chronic prostatitis) received totally 430 procedures of LBI. Each patient received 4-7 procedures of irradiation, exposition 30-40 min. each. HeNe and diode lasers (632.8 nm/2 mW at the end of the light-guide, inserted into a vein) were used for intravenous LBI. Diode IR lasers (890 nm/15 W pulse power, 3000 Hz, and 850 nm/200 mW CW laser) and HeNe 10 mW laser were used for transcutaneus LBI. Calculation of absorbed dosage was done. Each patient reported about sensations during and between procedures. The skin temperature changes were studied for patients with arterial occlusions of low extremities. After completing the therapy the comparison between results of treatment and sensations was done.
RESULTS
Different patients had different sensations, some others had no feelings. The typical sensations were warmth, reduction or elimination of pain, sleepiness during or after the procedure. Sensations depended on the diagnosis and condition of patients. It was shown, that 40% of angiological patients, 60% patients with trophic ulcers and septic wounds had sensations during the procedure. Such patients reported that sensations appeared at first in the mostly damaged area. Several patients with atherosclerosis of both low extremities mentioned that they had feeling of warmth in the worst affected leg. Later they reported that feeling of warmth had tendency to spread on larger areas and also appeared in the other leg (in relatively good condition). 90% of such patients reported, that sensations of warmth in every next procedure appeared earlier, were more expressed and covered larger areas than during previous procedure. In general the feeling of warmth lasted 30-90 min. after completing the procedure. Some patients reported several hour lasting warmth. Patients reporting sensations showed better treatment results. No patient with chronic prostatitis had sensations.
Temperature rising in areas of sensations during the procedure was registered among patients who had sensation of warmth.
Our studies proved that sensations associated with intravenous and transcutaneus infrared LBI were the same for 40% of patients. For other patients intravenous LBI caused earlier and more stressed sensations. To obtain the same sensations, transcutaneus IR LBI procedure had to be prolonged by 10-20%. No sensations during HeNe transcutaneus LBI were registered.
DISCUSSION
Sensations help to predict the effectiveness of the complex treatment. Those surgical and angiological patients who have no sensations will probably have more chances to have complications during the treatment. Future studies will find more correlation between results of instrumental investigations and sensations, reported by patients. The radiant power, absorbed by blood, is not enough to heat blood or organism. Probably sensation of warmth is caused by activation of local blood microcirculation.
The evaluation of sensations help to determine the individually required dosage of laser irradiation (duration of procedure, absorbed power of irradiation) more precisely. The best results of LBI were achieved if duration of each procedure was limited up to the point of maximal sensation. Probably it will be possible to determine the dosage of irradiation which will cause sensations among patients suffering from inflammatory disorders. The next step is to make treatment easier for patients, use non-invasive transcutaneus IR LBI procedures more widely instead of invasive intravenous version. Наверх
|
