Objective To summarize the recent progress in research on the mechanism of denerved skeletal muscle atrophy. Methods The recently-publ ished l iteratures at home and abroad on denerved skeletal muscle atrophy were reviewedand summarized. Results The mechanism of denerved skeletal muscle atrophy was very complex. At present, the studyof the mechanism was based on the changes in histology, cytology and molecules. Fiber thinning and disorderly arrangement of denerved skeletal muscles were observed and apoptotic bodies were detected. Apoptosis-promoting genes expressed upregulatedly and apoptosis-restraining genes expressed down-regulatedly. Muscle satell ite cells increased after denervation, but then they decreased and disappeared because they could not differentiate to mature muscle fibers. The structural change of cytomiscrosome and down-regulation of metabol ism-related enzymes induced cell metabol ism disorder. Conclusion The histological change of skeletal muscle fibers, the change of the number of muscle satell ite cells and differentiation, the structural change of cytomiscrosome and the change of apoptosis-related and metabol ism-related gene expressions contribute to denerved skeletal muscle atrophy.
OBJECTIVE: To investigate the effects of bone morphogenetic protein (BMP) on the proliferation and collagen synthesis of skeletal muscle satellite cells. METHODS: Skeletal muscle satellite cells were harvested and cultured in vitro. The 0 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml, and 1000 ng/ml BMP were used to induce skeletal muscle satellite cells for 48 hours. Cell proliferation, rate of myotube formation and collagen-1 synthesis were measured. RESULTS: BMP promoted cell proliferation and reduced the rate of myotube formation. Collagen synthesis increased when skeletal muscle satellite cells were induced with more than 500 ng/ml BMP. And the higher the concentration of BMP was, the ber this effect became. CONCLUSION: BMP can enhance the proliferation of skeletal muscle satellite cells and change their differentiation from myoblasts to osteoblasts.
In order to explore the effects of clenbuterol on intramuscular collagen metabolism in denervated skeletal muscles, a randomized, double-masked and placebo-controlled group were studied. Seventy-one patients with complete function loss in muscularcutaneous nerve resulted from brachial plexus injury were administered clenbuterol or placebo 60 micrograms Bid for more than 3 months. Biopsies of the biceps brachia muscle were performed at the beginning and end of this study. The biopsied muscles were processed with anti-collagen I and IV immunohistochemical stains and image analysis as well. The result showed that the collagen proliferation of both type I and IV was much reducible in the clenbuterol-treated group than that of the placebo-treated group (P lt; 0.05). It was concluded that clenbuterol could inhibit partially the proliferation of intramuscular collagens in denervated skeletal muscle.
Objective Extracellular matrix is one of the focus researches of the adi pose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sl iced into2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adi pose-derived stem cells (hADSCs) were isolated from adi pose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation abil ity. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Results Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibil ity. The CA and PI staining showed the seeded hADSCs had the potential of spread and prol iferation on the matrix. Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adi pose tissue engineering.
Objective To observe whether the motor nerve babysitter could improve the delayed nerve anastomosisand promote the functional recovery. Methods Sixteen SD rats weighing 200-250 g were randomly divided into 2 groups.In group A, the left musculocutaneous nerve was transected to make the model of biceps brachii denervation and anastomosed to its proximal end 6 weeks later; In group B, the musculocutaneous nerve was transected and the distal end was coapted to the purely motor medial pectoral nerve immediately (nerve babysitter) and the musculocutaneous nerve was separated from the medial pectoral nerve, and reanastomosed to its proximal end 6 weeks later. In the animal model, the left l imbs served as experimental sides, the right l imbs as control sides. After 6 and 12 weeks of the second surgery, behavioral test (grooming test) was performed and the degree of the biceps brachii atrophy was observed, the latent period and the ampl itude of the maximun action potentials of the biceps brachii were detected, the wet muscle weight, muscle fiber cross-section area and the activity of Na+-K+-ATPase of the biceps brachii were measured. Results After 4 weeks of the second surgery, grooming behavior was found in group B, while few grooming behavior was seen in group A till 6 weeks after the secondary surgery. After 6 weeks of the second surgery, the recovery rate of the latent period and the ampl itude, the wet muscle weight, muscle fiber cross-section area and the enzymatic activity of Na+-K+-ATPase of the biceps brachii in group A was 187.25% ± 1.97%, 46.25% ± 4.63%, 55.14% ± 1.99%, 49.97% ± 1.71%, and 65.81% ± 2.24%, respectively, which was significantly different from that in group B (155.96% ± 3.02%, 51.21% ± 2.13%, 74.18% ± 1.82%, 55.05% ± 1.64% and 71.08% ± 1.53%, respectively, P lt; 0.05). After 12 weeks of the second surgery, the recovery rate of the latent period and ampl itude, the wet muscle weight, muscle fiber cross-section area andthe enzymatic activity of Na+-K+- ATPase of the biceps brachii in group A was 145.36% ± 3.27%, 51.84% ± 5.02%, 77.92% ± 1.73%, 61.04% ± 2.68% and 71.94% ± 1.65%, respectively, which was significantly different from that in group B (129.83% ± 8.36%, 75.22% ± 2.78%, 84.51% ± 1.34%, 78.75% ± 3.69% and 84.86% ± 1.81%, respectively, P lt; 0.05). Conclusion Motor nerve babysitting could reduce muscular damage after denervation, improve the effect of delayed nerve repair and promote the functional recovery of musculocutaneous nerve.
Objective To investigate the influence of clenbuterol on the expression of nerve growth factor (NGF) in denervated red and white muscles and the neurotrophism of the denervated muscles.Methods Sixty-four Wister rats, weighed 200-250 g, were divided into 8 groups(8 rats per group), including 4 experimental groups and 4 control groups. The denervated model was made in rats by dissection of sciatic nerves. Clenbuterol was given at a dose of 200 μg/kg per day in the experimental group, saline in the control group. The expression of NGF was measured with immunohistochemistry after 1, 3, 7 and 14 days of injury. The culture methods of dorsal root ganglions of the chick embryos were used to measure the neurotrophism of extracts of the muscles. Results Compared with the control groups, the NGF content of gastrocnemious(GAS) increased on the 1st day (Plt;0.05) and the NGF content of soleus(SOL) increased greatly on the 1st, 3rd and 7th dayafter injury in the experimental groups (Plt;0.01). In the experimental groups, the NGF amount of GAS reached the highest value on the 1st day after injury(Plt;0.01) and then decreased gradually. And the NGF amount of SOL had slight difference between different time. The NGF content of the SOL was higher than that of GASon the 7th day (Plt;0.05). The sensory neurotrophism of the extracts was similar between SOL and GAS.Conclusion Clenbuterol can change the expression of NGF in denervated muscles, but the change was different in SOL and GAS. The sensory neurotrophism of the denervated muscles were determined by all of the neurotrophic factors in them.
In order to study the influence of reperfusion following ischemia on microvesseles and microcirculation of skeletal muscle, unilateral hindlimbs of 16 rabbits were subjected to normothermic ischemia for 2 and 5 hours by tourniquet. After release of the tourniquet, microcirculation of the peritenon on dorsum of the foot was observed for 1 hours by intravital microscope. At 1 hour and 72 hours following reperfusion, the anterior tibia muscle biopsiy were taken and the specimens were subjected to light and electron microscopic examinations. It was found that after release of the tourniquet, in the limbs undergone 2 hours ischemia, there was immediate and well distributed reflow in the microvesseles of peritenon though a few aggregates of red cells and increase in the number of adherent leukocytes occured in some venules, and the microvesseles of the skeletal muscle only showed signs of minimal injury, the muscle fibers could survive in the limbs undergone 5 hours of ischemia, however, there was serious disturbance of microcirculation in theperitenon, which was characterized by "no reflow" in most area and there was signi ficant increase in the number of leukocytes adherent to venular endothelium, and the microvesseles of the skeletal muscle showed signs of severe injury, including remarkable swelling of the endothelial cell, disruption of the basement membrane and interstitial edema, and finally, most of the muscle fibers had necrosis occured. The results demonstrated that reperfusion following ischimia might result in microvascular injury and microcirculation disorder in the ischemic area. The degree of the injury and disorder depended on the duration of ischemic period, and was an important factor which determined the fate of the parenchymal cell.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
OBJECTIVE To observe the ultrastructural changes and number of satellite cells in different muscles with different denervation interval and investigate the mechanism of denervation atrophy. METHODS Muscles of different denervation interval were harvested, which were 6 biceps brachii and 6 abductor digiti minimi. The ultrastructure of the samples were observed under transmission electron microscope. The number of nucleus and satellite cells were counted to calculate the percentage content of satellite cells. RESULTS In early stage of denervation, the myofilament and sarcomere of the majority were well oriented. The nucleoli of some muscle cell nucleus were enlarged and pale. Vacuolarization was also seen in some mitochondria. There was no obvious proliferation of collagen fiber around myofibers. After denervation of half a year, rupture and disorientation of myofilament was seen. The nucleus became smaller, dark stained, and some of them were condensed. There was proliferation of fibroblasts, adipose cells and collagen fibers around myofibers. Motor endplate was not recognized one year after denervation. In the early stage of denervation, satellite cell percentage of the two muscles was relatively high. It then declined with time. One year after denervation, satellite cells were scarcely detected. Comparison of the curves for satellite cell declination in two muscles revealed that the declination of the abductor digiti minimi was faster than that of biceps brachii. Decrease of the former started 3 months after denervation, while the latter started after 6 months. CONCLUSION Disappearing of motor endplate and proliferation of collagen fibers are main factors that affect the treatment outcome in late cases. Decrease of satellite cell number is another cause. The correlation of less satellite cell in abductor digiti minimi and poorer recovery of hand intrinsic muscles indicates that increment of satellite cells in long-term denervated muscles may be one of the effective measures to improve treatment outcome.
