[WEAK COMBINED MAGNETIC FIELDS ADJUSTED TO THE PARAMETRIC RESONANCE FOR Ca2+ INTENSIFY DYSTROPHIN SYNTHESIS IN MDX MICE SKELETAL MUSCLES AFTER CELL THERAPY].

The mdx mice are an X-linked myopathic mutants, an animal model for human Duchenne muscular dystrophy (DMD). Mdx mice muscles are characterized by high level of striated muscle fibers (SMF) death followed by regeneration. As a result most SMFs of mdx mice have centrally located nuclei. The possibility of using stem cells therapy for the correction of DMD is actively being studied. One of the approaches to the usage of bone marrow stem cells for cellular therapy of DMD is the replacement of bone marrow after irradiation by X-rays. This method however does not give significant increase of dystrophin synthesis in mdx mice muscles fibers. We have tried to affect the mice after bone marrow transplantation by weak combined magnetic fields adjusted to the parametric resonance for Ca2+(Ca(2+)-MF) based on the data that the weak combined magnetic fields influence on tissues regeneration. We observed a significant increase in the proportion of dystrophin-positive SMFs in group of mdx mice radiation chimera 5 Gy and 3 Gy which was additionally exposed in Ca(2+)-MF in comparison with the control mdx mice and the group of mdx mice radiation chimera 5 Gy and 3 Gy which was kept in terrestrial magnetic field 2 months after chimera preparation--up to 15.8 and 18.3%, respectively. Also, there was an accumulation of SMFs without central nuclei. These data indicate a significanly increased efficacy of cell therapy in the case of additional exposition in Ca(2+)-MF. Thus, the efficiency of bone marrow transplantation mdx mice after both in doses 3 and 5 Gy was considerably enhanced by additional exposition to Ca(2+)-MF. Apparently, such magnetic field can intensify functioning of donor's nuclei which had been incorporated into muscle fibers.

CHARACTERISTICS OF AMYLOID DEPOSITS DETECTED IN THE INTERNAL ORGANS OF mdx MICE.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

[Effect of bone marrow cells transplantation on the decidua formation in pseudopregnant rats].

One of the most common causes of the current pregnancy loss is the failure of the decidual reaction of endometrial cells. It is assumed that a partial source of decidual cells in endometrial tissue is bone marrow cells (BMCs). In the present work, we have studied possible effect of BMCs transplantation on the process of decidualization using the model of pseudopregnancy in rats. BMCs were flushed from the rat femurs and tibias. The obtained suspension of single BMCs was injected into one of rat uterine horns on the 5th day of pseudopregnancy. PBS without cells was injected into the contralateral horn served as the control. Rats were sacrificed on the 11th day of pseudopregnancy. Decidua formed in the experimental uterine horn showed an increase in the meso-antimezometral direction of their diameter of about 1.5-2 times as compared with a control horn. The weight of decidual tissue in the experimental horn exceeded 3 times the weight of the control one. The presence of transplanted BMCs in decidual tissue was documented by preliminary double staining of BMCs with membrane dye PKH 26 Red and nuclear dye Hoechst 33342. Histological analysis of decidua sections after transplantation revealed any alterations neither in cell differentiation nor in tissue structure. We conclude that BMCs transplantation stimulates decidualization in animals.

[Filling the 3D scaffolds by the stromal bone marrow cells depends on the methods of cell seeding and the scaffold surface modifications].

The distribution of bone marrow stromal cells (BMSC) is studied in a 3D poly(L,L-lactide) scaffolds. It is shown that the population of cells seeding into the scaffold with a peristaltic pump (dynamic method) allows the penetration of cells inside of the scaffold compared with the application of the cell suspension on the scaffold surface (static method). In contrast to cells seeding to scaffold by dynamic method the cells seeding by static method migrate from scaffolds in the first few days almost completely. It is found that BMSCs cultured in 3D polylactide scaffold modified by fibrin form colonies, while BMSCs cultured in 3D polylactide-scaffold modified by collagen type I distribute inside scaffold such single cells.

[The effect of NADP+ on electrophysiological properties of cardiomyocytes of C57BL/6 and mdx mice].

We have studied the influence of NADP+ on routine ECG in 6 months old C57BL/6 and mdx mice. The animals were anestheized by ether before ECG recording. ECG registration was made with the speed of 100 mm per sec. The first ECG recording was made before intraperitoneal NADP+ injection in a dose of 13 or 80 mg/kg. The second ECG recording was made in 10 min after NADP+ injection. Then anesthesia was cut off. The mice were occasionally anestheized 45-60 min later and the third ECG was recorded in 1 h after injection of NADP+. ECG recording was made at a speed of 100 mm/s in the standard leads I, II and III and in the unipolar leads AvR, AvL and AvF. Values of standard ECG characteristics such as the P wave, intervals PQ, QT, RR, and QRS complex in milliseconds were measured in standard lead II. We did not observe any differences between ECG magnitudes of 2-3 months old C57BL/6 and mdx mice during trial experiments. Mice of both lines had sinus rhythm of heart rate. QRS complex in mdx mice had a tendency to be larger compared with that in C57BL/6 mice. Heart rates fluctuated between 722 ± 22 and 681 ± 23 beats per minute. NADP+ influences was studied in 6 months old mice male. The increase in the RR interval and decreased heart rate from 697 ± 2 to 461 ± 23 and 491 ± 28 beats per min for C57BL/6 mice (P < 0.01) and from 722 ± 28 beats per minute to 454 ± 31 beats per min for mdx mice were registered in 10 min after NADP+ injection in a dose of 80 mg/kg. The increase in the RR interval can be explained by an increase in the interval QT. A statistically significant reduction in the QT interval leading to a decrease in the RR interval was observed in mdx mice in 1 h after NADP+ injection. NADP+ in a dose 13 mg/kg did not change mdx mice ECG properties significantly. ECG of mdx mice were characterized by negative repolarization of T wave in 37% between all leads. A deal of leads with the negative T wave repolarization decreased up to 3% in 1 h after NADP+ injection in a dose of 80 mg/kg. The results have shown that cytomembranes of ventricular cardiac myocyte and the degree of oxidative stress are the main touches of NADP+ influence in the cases of C57BL/6 and mdx mice hearts.

Mechanism of targeted migration of mesenchymal stem cells.

Experiments performed on mice C57Bl/6 with the use of GFP technology showed that activation of apoptosis is a potent physiological stimulus for targeted migration of mesenchymal stem cell from the blood to tissues. The effect of apoptosis-induced targeted migration of stem cells can be used in cell therapy of damaged organs and tissues in various pathological states.

Bone marrow stem cells repopulate thyroid in X-ray regeneration in mice.

The aim of this study was to clarify the regeneration mechanisms of thyroid in post-irradiated C57Bl/6 mice chimeric with transgenic green fluorescent protein (GFP) positive C57Bl/6 mice after 5 and 7.5Gy X-ray exposures with the aid of morphological and immunocytochemical research of GFP-positive cell distribution. Cryostat slides of larynxes with thyroid glands were fixed by mixture of cold methanol and ethanol, cell nuclei were stained with propidium iodide. After immunocytochemical staining the slides of larynx with thyroid gland were investigated by means of confocal LSM 5 PASCAL microscope. True GFP nature of green signals in tissue slides was confirmed via additional treatment by anti-GFP antibody and Texas Red labeled second antibody. Separate GFP-positive cells were observed in the walls of follicles and between follicles of chimeric mice 9-10 months after X-ray exposure. GFP signal was viewed as cytoplasmic droplets and within the colloid of follicles. The share of GFP-positive follicles reached 6.1±1.8%. There was also co-localization of GFP signals and positive staining for thyroglobulin by monoclonal antibody. As many as 20.8±1.8% among all propidium iodide positive blood cells and 52.3±8.3% among propidium iodide positive bone marrow cells were at the same time GFP-positive. In conclusion, the results show that the bone marrow stem cells participate in the thyroid gland regeneration after 5Gy X-ray exposure.

[NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation].

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.

[Effect of functional unloading and dystrophin deficit on the local hyperpolarization of the postsynaptic membrane of a skeletal muscle fiber].

The data have been obtained that confirm the identity of the electrogenic mechanism of hyperpolarization by nanomolar concentrations of cholinergic ligands in the extrasynaptic region and endogenous nonquantal acetylcholine in the synaptic region of a skeletal muscle fiber. In both cases, this mechanism is realized through the involvement of the alpha2 isoform of Na, K-ATPase and operates in the absence of Na+ entry through membrane channels. At the same time, there are peculiar properties which take place under functional disorders. Thus, the effectiveness of this mechanism in the synaptic region selectively increases under rat hindlimb unloading and decreases in case of dystrophin deficit in mdx mice. The last fact suggests that dystrophin is a molecular component that is essential for the functioning of the electrogenic mechanism of local hyperpolarization of the end-plate membrane.

[Effect of the impulse magnetic field on the growth bone plates in experimental animals].

The influence of impulse magnetic field (1.5 T) on growth bone plates of the rabbit has been studied. It has been found that this field produces the inhibitory action on the functional activity of growth bone plates.

[Structure of neuromuscular junctions and differentiation of striated muscle fibers of mdx mice after bone marrow stem cells therapy].

Mdx mice are a model of Duchenne muscular dystrophy caused by deficiency of dystrophin. Muscles of mdx mice are characterized by high levels of striated muscle fibers death and, accordingly, by a high level of its regeneration. Moreover, the structure of neuromuscular junctions in mdx mice is altered. Changes in the structure of mdx mice neuromuscular junctions against a background of increasing differentiation of striated muscle fibers after C57BL/6 Lin (-) bone marrow stem cells transplantation were investigated. The muscles were studied in 4, 8, 16 and 24 weeks after transplantation. We observed that the level of striated muscle fibers loss was decreased from the 4th week after transplantation of bone marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began from the 8th week, and dystrophin synthesis was increased at the 16th and 24th weeks after bone marrow stem cells transplantation. Longitudinal sections of quadriceps muscles of mdx mice showed decrease in the number of acetylcholine receptors clusters in neuromuscular junctions and a simultaneous increase in acetylcholine receptor clusters area during the 4th week after transplantation. In 16 weeks after bone marrow stem cells transplantation, total neuromuscular junction area was increased due to increase in the area of acetylcholine receptors clusters and to increase in their number as well. Thus, single intramuscular transplantation of C57BL/6 Lin (-) bone marrow stem cells induces an increase in differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.

[Characteristic of tumors developed after transplantation of transgenic GFP-positive C57BL/6 mice bone-marrow mesenchymal stem cells to mdx mice muscle].

The purpose of the study was the morphological and histochemical characteristics of differentiation of tumors developed after transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC) of transgenic mice C57BL/6 into M. quadriceps femoris of mdx mice. The tumors occurred only after transplantation of MSCs of 43-45th passages and did not arise after transplantation of MSCs of the 15th passage. No tumors developed also after transplantation of MSCs of 43-45th passages into muscle of C57BL/6 mice. The average weight of tumors appeared in 4 mdx mice studied was 1.3 +/- 0.5 g. All four tumors were classified as mesenchymomas because they originated from mesenchymal stem cells. Most of the periphery of the tumors was classified as fibrosarcomas with mitotic index 0.9 +/- 0.1%. The central parts of tumors had areas with epithelial like morphology of cells. Such cells showed positive reactivity for alcyan blue staining at pH 2.5, which indicated chondrocyte nature of the cells. No mitosis was observed in epithelial like cells. In the tumors, there were also areas with bone trabeculae containing megacaryocytes and foci of myeloid and erythrocyte hematopoiesis. There were also areas with neuronal and glial cells, and accumulations of adipocytes. One of the tumors was classified as a round cells sarcoma. The observed types of tumor cell differentiation in vivo were in accordance with described in literature types of MSCs differentiation after induction in vitro with special inductors. The spectrum of in vivo differentiation of transgenic GFP-positive MSCs after transplantation to mdx mice was broader than the spectrum of in vivo differentiation of transfected or transformed in vitro adult MSCs after transplantation to immunodeficient mice and mdx mice.

[Spontaneous transformation and immortalization of mesenchymal stem cells in vitro].

Mesenchymal stem cells (MXC) possess plasticity and unlimited proliferative activity in vitro that makes them an attractive subject of the studies focused on searching new resources for regenerative medicine. The usage of MSC is effective for treatment of patients with degenerative and traumatic diseases of different tissues, however, biological basis of therapeutic efficacy of MSC are still obscure. We found that long term culture of MSC expressing transgenic green fluorescence protein (GFP) led to increase in their proliferative activity, decrease in adhesion, and loss of differentiation potential and production of the GFP. MSC at the first passages showed karyotipic features of transformation, that at the later passages were complicated with developing of tumorigenic abilities detected after transplantation into normal syngenic recipients. When explanted into cell culture conditions the cells of the tumor tissue originated from the MSC did not express GFP and were not inducible to differentiation, but in contrast to the parent cells showed decreased clonogenic and proliferative activities. We suggest that growth of MSC in vitro results in their spontaneous transformation at early passages. Immortalization making physiological basis for the unlimited proliferation of MSC in vitro may be a feature of MSC transformation but not an initial characteristic of the stem cells.

[Cardiomyocyte survival and DNA repair in myocardium from C57Bl/6 and mdx mice after dynamical stress].

Mdx mice cardiomyocytes are a perspective model to study survival of terminally differentiated cardiomyocytes and formation of cardiomyopathy under conditions of oxidative stress. It was previously observed that dynamical stress induced formation of low molecular DNA fragments. It is beyond question that DNA fragmentation develops because of formation of double strand DNA breaks (DNA DSB). To record appearance and disappearance of DNA DSB we used antibodies to phosphorylated histone H2Ax (histone gamma-H2Ax.). The presence of DNA DSB was estimated in 0.05% and 6.7% of cardiomyocytes in the myocardium form C57B1 and mdx mice without stress, respectively. The part of cardiomyocytes with DNA DSB increased in an hour after stress up to 1.0% and 41.7% in C57B1 and mdx mice, respectively. In 24 h after stress, the myocardium from mdx mice contained 5.2% of gamma-H2Ax-positive cardiomyocytes and no C57B1 myocardium was found with any amount of gamma-H2Ax-positive cells. The results presented show induction of DNA damage by dynamical stress and restoration of normal DNA structure in the cells of both strains in 24 h after stress. There was no mdx mice death after used dynamical stress. To estimate the real contribution of DNA repair to the survival of cardiomyocytes we have counted the cardiomyocyte loss. Morphometric analysis demonstrated that cell concentration in myocardium from mdx mice under normal conditions was less than that one in myocardium of C57B1/6. The cell loss varied between 20% for the base and 40% for the apex of mdx mice hearts. In 24 h after stress, the cell loss in the myocardium of mdx mice amounted to 2.5%. The difference between the number of cells with damaged DNA structure and the index of the real cell loss allows concluding that DNA repair makes a real contribution to the survival of mdx mice cardiomyocytes after dynamical stress.

[Characterization of cultured murine mesenchymal stem cell line expressing GFP].

We have developed and characterized a murine mesenchymal stem cell line from the bone marrow of transgenic mouse C57BL ubiquitously expressing GFP. Immunostaining analysis revealed the presence of several markers typically found in fibroblasts such as smooth muscle cells actin in the form of stress fibrils and vimentin--the protein of intermediate filaments. These cells maintained capability to differentiate into adipocytes or osteoblasts under appropriate conditions. Karyotypic features include changes in the ploidy level between 2n and 8n and multiple chromosomal aberrations. After six passages 80% of the cell population was aneuploid with chromosomal numbers between 50 and 85 without well defined modal class. Differential G-staining of metaphase spreads showed variability in copy numbers of individual chromosomes and the presence of aberrations such as ectopic associations of non-homologous chromosomes. All analyzed cells contained unique dicentric marker chromosome and some of them also had numerous microchromosomes which might indicate gene amplification. These cells could be useful in the future work directed at the investigation of stem cells spontaneous oncogenic transformation in vivo and in vitro.

[Double strand DNA breaks in C57B1 and mdx mice cardiomyocytes after dynamical stress].

The survival of cardiac myocytes under different physiological and pathological conditions presents pressing problem. mdx mice cardiac myocytes are a promising model of cell survival under condition of oxidative stress. Our early results have shown that some part of mdx mice cardiomyocytes is in early stage of apoptosis (Kazakov, Mikhailov, 2001). But the development of cell death with loss of apoptotical cardiac myocytes occurs only after dynamical stress (bathing during 5 min) (Mikhailov et al., 2001). DNA endonuclease activity in the myocardium and low level of cardiac myocytes death during usual being of mdx mice allowed us to suggest DNA repair to be involved in the survival of mdx mice cardiac myocytes (Mikhailov et al., 2003). To confirm the suggestion we have studied the dynamics of formation and elimination of double strand DNA breaks in mdx myocardium cells after 5 min bathing at 12 degrees C. To visualise double strand DNA breaks formation cell nuclei were stained by monoclonal antibodies to phosphorylated H2Ax histone and to mouse PAP. Double staining with monoclonal anti-H2Ax antibodies and monoclonal anti-a-actin antibodies were used to separate cardiac myocytes from other myocardial cell types. The results showed that during 40 min after stress the deal of H2Ax-positive nuclei in mdx myocardium cells grew up to 41.7 +/- 11.4 % as compared with the initial control level of 6.7 +/- 0.2 %. The number of H2Ax-positive nuclei in these cells decreased after 24 h to 5.7 +/- 0.2 %. The quantity of tagged myocardium cell nuclei in C57B1/6 mice after stress was negligible and did not go beyond 0.01%. Dynamical stress also induced the increase in the rate of 3H-Thymidine incorporation by mdx mice cardiac myocytes from 0.3 +/- 0.3 up to 2.9 +/- 0.5 %. There was not change in the rate of 3H-Thymidine incorporation by cardiac myocytes in C57B1/6 mice. The numbers of labelled nuclei before and after stress were 0.2 and 0.3 %, correspondingly. The number of 3H-Thymidine labelled mdx cardiac myocytes fell down up to 0.4 +/- 0.2 % within 24 h after stress; the level of labelled C57B1/6 cardiac myocytes did not change. We have concluded that 3H-Thymidine incorporation into cardiac myocytes nuclei and staining of these nuclei by monoclonal antiboies phosphorylated H2Ax histone after stress demonstrate rather DNA repair than cardiomyocytes entry into the cell cycle.

[Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle].

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.

[Quantitative assessment of current functional state under stressful conditions].

Spectral analysis of heart rate variability was used for quantitative assessment of human functional state (FS) under stressful conditions. The results of the investigation of three groups consisting of apparently healthy individuals such as candidates for becoming cosmonauts (1), sportsmen-parachutists (2) and sportsmen of cyclic kinds of sports (3) demonstrate that there are common patterns of response to stress. In all the groups the decrease of general spectral power at the expense of sympathetic (LF) and, to greater extent, parasympathetic (HF) components was detected. In the 1st and the 2nd groups it was associated with significant increase of the ratio LF/HF and the reduction of parasympathetic reactivity (the coefficient made up 30/ 15). However, in contrast to the 1st and the 2nd groups, such changes were lacking in the 3rd group. It has been concluded that the results of this investigation may be taken into account in the course of the assessment of human FS under stressful conditions. The ways of their interpretation depending on spectral characteristics are given.