THE HISTOPATHOLOGICAL CHANGE AND DYSTROPHIN EXPRESSION IN THE MDX MICE BY BONE MARROW CELL TRANSPLANTATION
C. Zhang*; W. Zhang; Z. Liu; X. Lu; X. Wang
Dept Neurol, First Affiliated Hosp, Guangzhou, China
[Objective] To observe the histopathological change and dystrophin expression in the mdx mice by bone marrow cell transplantation.
[Methods] Different amounts of bone marrow cells, suspension cells and stromal cells were cultured in vitro from C57BL male mice, and the cells were transplanted intravenously into the 14 irradiated (8 Gy) mdx mice . The survival rate and survival time were investigated. 1, 2, 3, and 4 months after transplantation, the muscle histopathological analysis was assessed with immunofluorescent technique. Sex determined region of the Y chromosome from the transplantation female mdx mice were examined by means of PCR.
[Results] 3 months after transplantation, 10 mice were alive, and 4 mice died. A few dystrophin was observed in the mdx mice muscle about 1 or 2 months after transplantation. By 3 months after bone marrow cells, suspension cells, and stromal cells transplantation, about 10 %, 8 % and 6 % of the muscle fiber expressed dystrophin respectively. About 449 bp DNA fragments from Y chromosome were amplified in the female mdx mice.
[Conclusion] This study shows that most mdx mice can survive by being transplanted bone marrow cells, suspension cells, and stromal cells. Dystrophin can be expressed by different component of marrow cell transplantation, and the expression time of dystrophin can keep on at least over three months.
Supported by the National Nature Science foundation of China (39870804), Guangdong Nature Science foundation (970061)

 

MANGANESE QUENCH RATES AND STEADY STATE CALCIUM CONCENTRATIONS IN CULTURED DYSTROPHIC MYOTUBES AND FRESHLY EXCISED ADULT DYSTROPHIC FIBERS.
C.G. Carlson*; R. Ashmore; A. Gueorguiev; D. Roshek; J. Chu
Dept. of Physiology, Kirksville College of Osteopathic Medicine, Kirksville, MO, USA
To examine the potential role of elevated intracellular [Ca] and enhanced resting Ca influx (RCI) in the pathogenesis of myopathies, steady state [Ca] was estimated from F340/F380 ratios and known [Ca] standards, and RCI was estimated from the rate of Mn quenching of intracellular signal in both FURA PE3 loaded spontaneously inactive cultured myotubes and freshly excised triangularis sterni (TS) muscle fibers. Initial studies indicated a significant effect of genotype on both F340/F380 ratios (p<0.05) and Mn quench rates (p<0.05), with utrophin-mdx double knockout (DKO) myotubes exhibiting a [Ca] (59.9 nM, 10.2SEM) that was about 68% larger than nondystrophic and 123% larger than mdx myotubes. In the presence of 100 microM ACh, both mdx and DKO myotubes had elevated Mn (1 mM) quench rates with DKO myotubes having rates (25.9 x 10(-4) sec(-1)) that were about 2.5 fold larger than mdx myotubes. Adult mdx TS muscle fibers, which are severely dystrophic, had significantly (p<0.001) elevated F340/F380 ratios and an estimated mean [Ca] of 218 nM in comparison to 113 nM in nondystrophic fibers. In the presence of Ca, the Mn (0.5 mM) quench rate in mdx TS fibers was elevated in comparison to nondystrophic fibers, but the difference failed to reach statistical significance (p>0.05). Quench rates (1.5 mM Mn) obtained in the absence of Ca were identical between nondystrophic and mdx TS fibers (2.2 x 10(-4) sec(-1)). These results are consistent with the general hypothesis that elevated steady state Ca concentrations promote the destruction of muscle fibers in myopathies.
Supported by: an SRI award from KCOM.
NEURONAL POTENTIAL OF STEM CELLS FROM ADULT MUSCLE.
M. Romero-Ramos1*; Y. Wu1; R. Zaman1; P.A. Lucas2; H.E. Young3; M.-F. Chesselet1
1. Dept of Neurology, UCLA School of Medicine, Los Angeles, CA, USA
2. Dept of Orthopedic Surgery, New York Medical Collage, Valhalla, NY, USA
3. Div of Basic Medical Science, Mercer University School of Medicine, Macon, GA, USA
Stem cells have been identified within the connective tissues of skeletal muscle (Lucas et al. 1995). These cells can differentiate into multiple phenotypes of the mesenchymal and other lineages (Lucas, Young, unpublished observations), suggesting that they are multipotent stem cells. It is unclear, however, whether these cells could be induced to primarily express a neural phenotype. We have isolated stem cells from adult rat muscle and exposed them to growth factors and defined differentiating conditions for 5 and 24 hours. Cells were examined with immunocytochemistry and fluorescence assisted cell sorting. At 5 hours, more than 90% of the cells co-expressed phenotypic markers of mature neurons and glial cells. This pattern of co-expression has been previously shown in neuro-glial progenitors, suggesting that stem cells from adult muscle can differentiate along a neuroectodermal lineage. At 24 hours, most cells still expressed neuronal markers whereas only a subset co-expressed glial markers. Our study confirms the presence of multipotent stem cells in adult muscle and shows that these cells are able to overcome germ lineage restriction and express the characteristics of neural stem cells. Therefore, stem cells isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative diseases.
Supported by: a BioStar Grant, P50-NS 38367, The Pasarow Foundation and MorphoGen Pharmaceuticals, Inc.
EFFECTS OF NEUROPEPTIDE Y (NPY) ON MUSCLE PERFORMANCE AND FATIGABILITY IN RAT ISCHEMIC MUSCLES
I. Kalezic1; E.W. Lee2; M. Ljubisavlijevic1; H. Johansson1; Z. Zukowska1,2*
1. Centre for Musculoskeletal Research, NIWL, Umea, Sweden
2. Physiology & Biophysics, Georgetown Univ., Washington, DC, USA
Recently, Zukowskas group has shown that NPY, a sympathetic neurotransmitter and a vasoconstrictor, is angiogenic and induces revascularization of the rat ischemic limb. Using this model, we now studied the performance and fatigability of the gastrocnemius muscle under isometric conditions at 14 days after femoral artery occlusioninduced ischemia and local administration of NPY (1mg/14 days/pellet, IsN rats) or placebo (IsP), and compared to control non-ischemic muscles (C). The gastrocnemius muscle was unilaterally stimulated via the lateral and medial gastrocnemius nerve or corresponding filaments isolated from L5 and L6 ventral root using repetitive tetanic stimulation (600 ms duration, 85 Hz fusion frequency, 1 sec intervals). Series of increasing number of trains (1,3, 6, 12, 30 and 60, 60 sec intervals) were applied to each muscle. Current intensity was 3-5 times the motor threshold in order to evoke synchronous activation of all muscle fibers. During trains 1-6, relative forces dropped faster in IsP than in IsN and C muscles, while during trains 12-60, tension diminished more in IsN and C. In IsP muscles, there was a rapid, brief increase in tension followed by a quick drop to 1/3 of the peak value, which remained stable during the whole train, as compared to C and IsN muscles where a fused tetanus occured. These data suggest that NPY preserves the ability of different fast-twitch fiber populations to produce submaximal tetanic force in isometric condition, and restores muscle performance and fatigability altered by ischemia.
Supported by: Swedish Work Life Res. Council & HL67357