Use of myogenic cells for the treatment of Duchenne’s disease was first suggested by Partridge et al. [1]. The development of the genetically authentic model of this desease – mdx mice allowed to test myoblasts and other types of stem cells in the therapy of this pathology [1]. In the experiments with GFP-marked cells it was shown that bone marrow (BM) stem cells take part in the regeneration of mdx mice striated muscle fibres (SMF) [2-6]. Our attention to this type of stem cells due to the data that BM, being the source of stem cells plays the leading part in the system of mammalian stem cells [7].

In the skeletal muscle stem cell niches of wild type mice as well as of the mdx mice [8] are sites for BM stem cells differentiation into satellite cells and other types of muscle stem cells [8-11] [10]. It is essential to say that cells expressing the hematopoietic marker CD45 are predominantly present in the BM but also reside in the skeletal muscle and participate in muscle repair [12,13] and in muscle of mdx mice after BM stem cells transplantation [6].

Except blood and muscle cells BM stem cells are also involved in the cell differentiation and regeneration in lungs, liver, skin, gastrointestinal tract and of epithelium of thyroid gland [6,14,15].

Irradiation is a mandatory preliminary step of BM stem cells transplantation in recipient, as it suppresses the immune system of the recipient, making it possible to replace mutant bone marrow with the bone marrow of wild-type. The aim of this work was to study the recovery of dystrophin synthesis after C57BL/6 mice BM transplantation in mdx mice irradiated at a non-lethal dose of 3 Gy [28,29]. To estimate the significance of change of dystrophin synthesis for physiology of striated muscles there was study of structure and function of nerve-muscle junctions of diaphragm muscle fibers.

Animals

C57BL/6 mice have been received from Rappolovo animal farm (St. Petersburg, Russia). mdx mice were provided by Professor Partridge (Hammerssmith Hospital, UK) [1]. Mice were maintained in animal nursery (Institute of Cytology RAS) with standard feeding and light regimes.

Transplantation

1-1.5 months old mdx mice were irradiated by x-ray at a dose of 3 Gy using roentgen device rum-17 (Russia) using 0.5 mm CU + 1.0 Alu filters. Energy of X-ray was 45 R/min. Next day freshly prepared bone marrow (BM) cells were injected intravenously in dose (15-20) x 10⁶ cells per mouse. Animals were studied through 2, 4, and 6 months after transplantation. Each experimental group of mice included 3-6 animals. Mus. quadriceps femoris and diaphragm muscle fibers with their nerve-muscle junctions (NMJs) were under investigation.

For chimerism registration a special study was conducted with transplantation of GFP-positive BM cells, friendly provided by Dr. V. Serikov (CHORI, Okland, USA) after 3 Gy irradiation. At 6 month after transplantation BM cells were separated from long bones and smears were prepared by propidium iodide and studied on confocal microscope LSM 5 Pascal (Carl Zeiss, Germany) to count the part of GFP-positive cells to whole quantity of cells. There were used 8 C57BL/6 and 45 mdx mice.

Immunohistochemistry

10 μm cryosections were cut with cryostat (Bright Co, Ltd, UK) after preliminary muscle freezing in liquid nitrogen. Dried sections were fixed in ethanol mixed with carbinol (1:1 v/v) for 1 min at -20°C or in 10% formalin for 30 minutes at room temperature [30].

For dystrophin staining sections fixed in ethanol/carbinol (1:1 v/v) solution were treated with 1% bovine serum albumin (BSA) for 30 min, washed with PBS for 5 min and incubated with rabbit polyclonal antibodies to dystrophin (Abcam, US) dilution 1 : 100 for 1 h. Samples were washed with FITS-labeled goat anti-rabbit antibody (Sigma, US), 1:150, 1 h. Sections washed with DAPI or propidium iodide, were mounted in glycerol, and assayed under LSM 5 Pascal microscope (Carl Zeiss, Germany). The number of SMFs with dystrophin and fraction of dystrophin-positive fibers in central muscle were counted on sections.

To estimate the number of dead SMFs and SMFs without centrally located nuclei sections were stained with hematoxylin-eosine, passed through ethanol and xylol grades and mounted in Canada balsam. Stained sections were visualized under Axiophot microscope (Carl Zeiss, Germany).

NMJs assessment

Muscle longitudinal and cross sections fixed with 10% formalin were treated with 1 μg/ml tetramethylrhodamine-α-bungarotoxin (TMR-α-BTX) (Biotium, US) for 1 h. Then the sections were washed three times with PBS for 5 min, mounted into reagent that reduced unspecific fluorescence (Biomeda Co, US) and visualized under LSM 5 Pascal (Carl Zeiss, Germany).

NMJ structure was examined in records of single NMJ. NMJ area was calculated on muscle cross sections with ImageJ software (National Institute of Health, US). The area of single acetyl-choline receptor (AChR) clusters that makes the NMJ and the number of AChR clusters in each NMJ were estimated on the longitudinal sections with ImageJ software. The area of each NMJ includes area of AChR and areas of gaps between single clusters. The data were statistically processed with Microsoft Excel. The differences between groups were assessed using Student’s t-test. The differences were considered to be significant at p < 0.05 [30].

Membrane potential recording

The experiments were performed on freshly isolated diaphragm muscle as described previously [31,32]. A diaphragm strip with nerve stump was placed in a Plexiglas chamber. The chamber was continuously perfused with a physiological solution containing (mM): NaCl, 137; KCl, 5; CaCl₂, 2; MgCl₂, 2; NaHCO₃, 24; NaH₂PO₄, 1; glucose, 11; pH 7.4. The solution was continuously bubbled with 95% O₂ and 5% CO₂ gas mixture and maintained at 28°C. The muscle was equilibrated for 1 hour prior to the start of recording. The resting membrane potential (RMP) were recorded intracellularly using standard microelectrode techniques. Recordings were made in extrajunctional membrane regions within 1–2 mm from visually identified terminal branches of the nerve, and directly at the nerve terminal (end-plate region, junctional membrane) of the same muscle. RMPs were recorded from 15 to 25 different fibers within each muscle. The entire protocol was repeated in muscles from different animals to obtain the average resting potential for that condition. Data are given as the mean ± SEM. Statistical significance of the difference between group’s means was evaluated using a Student’s t-test (ORIGIN 6.1. software).

There was an accumulation of dystrophin positive mdx mice SMF through 2 months after syngenic bone marrow transplantation up to 4.1% in compare with 1.1% of control animals. Dystrophin synthesis accelerates 2 times for every 2 months and reach 27.7 ± 6.7% at 6 months after transplantation (Table 1 and 2) (Figure 1). We observed also the duplication of SMF without central nuclei up to 22.6% and loss of dead SMF up to 0.7 ± 0.1%. The obtained level of dystrophin synthesis (27.7 ± 6.7%) is consistent with data of other authors concerning 20% level of dystrophin-positive SMF as critical minimal working level for the function of dystrophin-deficient muscles [21,22]. Also we observed 11.2 ± 3.3% of dystrophin-positive SMF in diaphragm in four months after transplantation (Table 2) which is consistent with data obtained from skeletal muscle. The part of GFP-positive cells between all cells with nuclear on the BM smears of chimeric GFP transplanted mdx mice at 6 months after transplantation was 3.3 ± 0.8 % that indicates the chimeric nature of mice. All results point to the increase of differentiation level of mdx mice SMF after change of mutant bone marrow for bone marrow of wild type.

Table 1: Dystrophin synthesis by SMF of mdx mice M. quadriceps femoris after x-ray irradiation at a dose of 3 Gy followed by BMC transplantation (%, X ± m_x).

Figure 1: Transverse sections of M. quadriceps femoris of control С57BL/6 mice (a), of control mdx mice (b), and of chimeric mdx mice after 3 Gy irradiation and 6 months BMSCs transplantation (c). Immunostaining for dystrophin with biotin labeled second antibody and streptoavidin with peroxidase targeted. Nuclei were observed by Gimsa staining. Mag. Ob.10x, Oc.10x. Bar 60 mkm.

Study of NMJs structure and their resting potential was conducted for two reasons. We have shown early that local transplantation of BM stem cells to M. quadriceps femoris of unirradiated mdx mice partly restore the structure of NMJs [30]. To confirm the results we studied a structure of diaphragm NMJs on the transverse section of synapsis region of diaphragm SMFs by staning with Tetramethylrhodamine-α- bungarotoxin after general transplantation of BM cells. The synapsis of diaphragm mdx mice are formed by islets (75.8%) and branches (14%). In four months after BM transplantation we observed a significant increase in quantity of branches (43 %) and decrease in the part of islets in the diaphragm NMJs up to 53.7 ± 6.0 % (Figure 2 and Table 3). Development of branches and decrease of islets are considered to be the markers of the fractional reparation of NMJs structure. In case of local transplantation of BMSCs we did not observed the formation of branches. High concentration of islets and growth of square of NMJs taken place on the background of negligible dystrophin synthesis near 2% [30]. To estimate functional significance of the structural changes of diaphragm NMJs we studied the electrophysiological properties of NMJs through 4 months after BM transplantation (Table 4).

Table 3: Reparation of structure of diaphragm NMJs after x-ray irradiation at a dose of 3 Gy and bone marrow transplantation.

Table 4: Reparation of the resting membrane potential of mdx mice diaphragm muscle after bone marrow cells transplantation.

Figure 2: A. Diaphragm of C57BL/6 mice. Red –acetylcholine receptors; green – neurofilaments, NMJ consists from acetylcholine receptors organized as branches; bar -25 mkm B. Diaphragm of mdx mice. Red colour – acethylcholine receptors, green colour – neurofilaments, NMJ is consists from acetylcholine receptors organized and formed by islets.

In the control C57B1/6 mice, the value of resting membrane potential (RMP) in the junctional (end-plate) region of NMJs was –81.4 ± 0.5 mV being more negative than that in the extrajunctional region of sarcolemma (–78.0 ± 0.4 mV) (p < 0.01). The value of the observed local hyperpolarization corresponds well to the previous results and is specific for intact muscle fibers [28,31]. In mdx mice the value of RMP was lower than in the control C57B1/6 mice. The observed depolarization is in agreement with the well-known facts [33]. Moreover, the values of RMP through overall membrane did not differ. However, in mdx mice after 4 months of BM cells transplantation, the values of RMP in the junctional and extrajunctional regions increased to the level of control C57B1/6 mice. In the end-plate region, the value of RMP increased by 5.6 mV while in the extrajunctional region, it increased by 2.6 mV. As a result, local hyperpolarization of the endplate increased by 3.7 ± 0.9 mV (p < 0.01), which was characteristic of the control animals and is a reliable indicator of a normal functioning of a neuromuscular synapse after BM transplantation (Table 4).

Originally our choice for the dose of 3 Gy for irradiation was triggered by Abedi et al. [5] and our results [6], as it was impossible to get high stable level of dystrophin synthesis by mdx mice SMF by varying lethal doses of X-ray irradiation. Early we described the positive influence of 3 Gy irradiation and change of mutant BM for bone marrow of wild type for dystrophin synthesis by SMF [28,29]. X-ray irradiation doses between 1.5 and 3 Gy induce the acquisition of stable mixed chimerism in recipients. Such type of nonmyeloablative stem cells transplantation permits rapid engraftment from sibling and related donors with minimal toxicity, induce stable mixed chimerism and donor specific transplantation tolerance [34,35]. There are also some descriptions of successful cure of patients with nonmalignant hematologic diseases and congenital immunodeficiencies [36,37]. Level of figures of chimerism depends on used methods. Low range of chimerism in our experiment (3.3 %) may be explained by imperfection of used methods of chimerism determination in comparison with results of other authors. In case of successful allo-BM transplantation of patient with Diamond-Blackfan anemia and Duchenne muscular dystrophy the mixed chimerism was observed with 8-10.4 % donor cells in the muscle biopsy too [38].

Our results demonstrates that transplantation of wild type BM cells into mdx mice after 3 Gy irradiation is more effective for dystrophin synthesis reparation then after irradiation at a dose of 5 Gy or higher. The growth of dystrophin was observed in ?. quadriceps femoris and in SMF of diaphragm. Through 6 months the part of dystrophin positive SMF reached 27.6 ± 6.7 % which is a critical minimal working level for the function of dystrophin-deficient muscles (20%, [21,22]). Practically it means the cure of mdx mice after BM transplantation.

There are several reasons that may explain the success of BM cells transplantation after 3 Gy irradiation. We speculate that except suppression of immunological conflict the suppression of dystrophin synthesis after irradiation in lethal doses and bone marrow stem cells transplantation is caused by the disturbance of SMF sarcoplasm molecular systems that take part in regulation of nuclei differentiation of transplanted SMFs. Moreover, mdx mice have an altered expression level of 1735 genes from the studied 7776 genes, including genes of Notch-Delta and Neuregulin 3 signaling pathways that cause activation, proliferation and differentiation of satellite cells [39]. There is no doubt that after the radiation exposure in mdx mice at a lethal dose of 5 Gy or higher the disturbance of this signal paths increase even more. In our experiments the dose of 5 Gy was lethal for mdx mice [6].

We believe that the reparation of NMJs structure after BM transplantation, as well as dystrophin synthesis restoring, are the evidences of effectiveness of BM exchange in nonmyeablative bone marrow transplantation. We suggest that positive effects of BM cells exchange are connected with dystrophin synthesis. Local transplantation of Lin(-) BM cells in M. quadriceps femoris of mdx mice aggregates islets of bungarothoxin-positive substance for large NMJs but without branches. In this case the level of dystrophin synthesis did not exceed 2% [30]. The more extensive level of dystrophin synthesis in case of whole body irradiation 3 Gy followed by BM cells transplantation (Table 2) correlates with reparation of NMJs and branches enrichment. Our results support the conclusions of Kong and Anderson [40] and Banks et al. [41] for the importance of dystrophin participation in NMJs formation.

To confirm the nonrandomness of structural changes a study of electrophysiological properties of NMJs was conducted. We observed reparation of resting membrane potential of diaphragm muscle after BM exchange. Value of local hyperpolarization of the end-plate reached 3.7 ± 0.9 mV. It is typical for the wild-type animals and is a reliable indicator of normal functioning of a neuromuscular synapse [42,43] after BM transplantation (Table 4).

The use of of nonmyeablative bone marrow transplantation may be a next step in the cure of mdx mice and recipients with Duchenne muscular dystrophy disease.

The study was made with financial support of MCB program of RAS, of St. Petersburg’s State University Research Programm ? 1.37.118.2011 and by RFBR Grant ? 10-04-00970a.