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.
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 investigate the pathological changes in the neuromuscular junction during ischemiareperfusion(IR) in the skeletal muscle. Methods Forty-eight healthy adult Wistar rats (24 male, 24 female) were equally randomised into the following 6 groups: Group A (control group): no ischemiareperfusion; Group B: ischemia by clamping the blood vessels of the right hindlimb for 3 hours; Group C: ischemia by clamping for 4.5 hours;Group D: ischemia by the clamping for 4.5 hours followed by reperfusion for 1.5hours; Group E: ischemia for 4.5 hours followed by reperfusion for 24 hours; and Group F: ischemia for 4.5 hours followed by reperfusion for 2 weeks. Then, the medial head of the gastrocnemius muscle flap model was applied to the right hindlimb of each rat. The medial head of the gastrocnemius muscle was isolated completely,leaving only the major vascular pedicle, nerve and tendons intact.The proximal and distal ends (tendons) were ligated while the vessel pedicle was clamped. And then, Parameters of the muscle (performance,contraction index,colour,edema,bleeding) were observed. The muscle harvested was stained with gold chloride(AuCl3) and the enzymhistochemistry assay (succinate dehydrogenase combined with acetylcholine esterase) was performed. Morphology and configuration of the neuromuscular junction were observed during the ischemiareperfusion injury by means of the AuCl-3 staining. The result of the enzymhistochemical reactions was quantitatively analyzed with the computer imageanalysis system. And then, additional 5 rats were prepared for 3 different models identical with those in Groups A, C and E separately. The specimens were harvested from each rat and were stained with HE and AuCl-3, and they were examined under the light microscope. Results During the period of ischemia, the skeletal muscle of Group B showed the colour of purple and edema.The colour and edema became worse in Group ,while dysfunction of elasticity and contraction appeared obviously with plenty of dark red hemorrhagic effusion at the same time.After reperfusion,the color and edema of muscle in Group D became improved while the elasticity and function of contraction was not improved. Hemorrhagic effusion of Group D turned clearer and less than Group C.Group E was similar to Group D in these aspects of muscle except for much less hemorrhagic effusion. Skeletal muscle in Group F showed colour of red alternating with white, adhesion,contracture of muscle, exposure of necrotic yellow tissue and almost lost all its functions. The AuCl3 staining showed that during IR, necrosis of the myocytes was followed by degeneration of their neuromuscular junctions, and finally the nerve fibers attached to these neuromuscular junctions were disrupted like the withering of leaves. The enzymhistochemistry assay showed thatthere was no significant difference in the level of acetylcholine esterase between the ischemic group (Groups B and C) and the control group (Group A) (Pgt;0.05). However, the level of acetylcholine esterase in all the reperfused groups (Groups D, E and F) decreased significantly when compared with the control group（Group A）and the ischemic groups (Groups B and C) (Plt;0.01). Conclusion The distribution of the nerve fibers and the neuromuscular junctions in the mass of the muscles is almost like the shape of a tree. The neuromuscular junction seems to be more tolerant for ischemia than the myocyte. Survival ofthe neuromuscular junction depends on its myocytes alive. Therefore, an ischemiareperfusion injury will not be controlled unless an extensive debridement of the necrotic muscle is performed.
Objective To review researches of the role of inhibitorof differentiation 2(Id2) in skeletal muscle regeneration. Methods The latest original literature concerning Id2 and its role in skeletal muscle regeneration was extensively reviewed. Results Id2 could form heterodimers by combining with E protein to prevent myogenic regulatory factors (MRFs) forming heterodimers by combining with E protein, to inhibit the transcription activity of MRFs anddifferentiation of skeletal muscle cell. Conclusion Id2 plays an important role in skeletal muscle regeneration.
Objective To study the protective effects of ischemic preconditioning(IP) duration against ischemic reperfusion injury of skeletal muscle. Methods Thirty-six Wister rats were made amputation-like models, which underwent temporary amputation at the level of the femur, excluding the femoral vessels. They were divided into 6 groups(n=6) according to different treatments before ischemiareperfusion: group A(4 hours of ischemiareperfusion); groups B, C, D, E(5, 10,15, 20 minutes of ischemia and 5, 10, 15, 20 minutes of reperfusion respectively, for 3 cycles, 4 hours ischemiareperfusion ); group F (no ischemia-reperfusion). The malondialdehyde(MDA), the extent of edema and necrosis of skeletal muscle were measured to observe protective effects of different ischemic preconditioning duration. Results Five minutes of ischemic preconditioning（IP5）could protect skeletal muscle of ischaemia against necrosis and the survival area of the muscle was 82.47%.The effects of IP10 and IP 15 were significantly superior to that of IP5 and the survival areas of the muscle were 89.03% and 89.49%. The effect of IP20(78.27%) was significantly inferior to that IP5. IP5 could reduce edema of skeletal muscle, the effect of IP10 was significantly superior to that of IP5. IP5, IP 10,and IP 15 could decrease the level of MDA, but IP20 did not decrease it. Conclusion The trend of protective effect of IP on ischemia-reperfusion injury of themuscle in rats first rise to the peak and then go down,10minutes ofIPis optimal.
OBJECTIVE: To study the influence of the electric stimulation of denervated muscle atrophy. METHODS: Sixteen SD rats were made the model of denervated skeletal muscle in two lower limbs by cutting off the sciatic nerve and femoral nerve. The right gastrocnemius muscle was stimulated with JNR-II nerve amp; muscle recovery instrument by skin as the experimental side and the left was not treated as the control side. The muscle histology, ultrastructure, fibrillation potential amplitude, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activities were observed 2 weeks and 4 weeks after operation. RESULTS: Electric stimulation could protect mitochondria and sarcoplasmic reticulum from the degeneration. The reduction rates of muscle cell diameter and cross section in the experimental side were slower significantly than those in control side. There was no influence on fibrillation potential amplitude in the both sides after electric stimulation. The reduction rates of Na(+)-K(+)-ATPase activity in the experimental side were slower 15.59% and 27.38% respectively than those in the control side. The reduction rates of Ca(2+)-ATPase activity in the experimental side were slower 4.83% and 21.64% respectively than those in the control side. CONCLUSION: The electric stimulation can protect muscle histology, electrophysiology and enzymic histochemistry of denervated skeletal muscle from the degeneration. The electric stimulation is an effective method to prevent and treat muscle atrophy.
Objective To explore the effect of basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）on the growth of muscle derived stem cells(MDSCs). Methods MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal musclecell marker αSarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25,12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24,48,72 and 96 h.Results MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to α Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration.bFGF began to show significant proliferative effect at 12.50 ng/ml (P＜0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P＜0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with thatat 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (Plt;0.01). When they were applied together, significant effect was shownat 24 h (Plt;0.01) and much higher effect was observed at 48, 72 and 96 h (Plt;0.05). Conclusion Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and ber.
Objective To investigate the role of AKT/FOXOs /atrogin-1/MuRF1 signaling pathway in skeletal muscle atrophy in rats with chronic obstructive pulmonary diseases( COPD) .Methods Passive cigarette smoking was used to establish COPD model. The protein expression of atrogin-1, MuRF1, FOXO-1, phosohorylated-AKT and total AKT were measured by Western blot. The mRNA expression of atrogin-1, MuRF1 and FOXO-1 were measured by reverse transcription-polymerase chain reaction( RT-PCR) . Results Compared with the control group, the mRNA expressions of atrogin-1, MuRF1 and FOXO-1 significantly increased in extensor digitorum longus ( EDL) of the COPD group (Plt;0.05 ) . Meanwhile the protein expression of atrogin-1 and MuRF1 significantly increased in the COPD group(Plt;0.05) , while the protein expression of FOXO-1 was not significantly different between two groups(Pgt;0.05) . In addition, , the protein expression of phosohorylated-AKTand the ratio of phosohorylated-AKT to total AKT significantly increased in EDL of the COPD group(Plt;0.05) . Conclusion The mRNA and protein expression of AKT/FOXOs/ atrogin-1 /MuRF1 in skeletal muscle are significantly increased in COPD rats, suggesting that AKT/FOXOs/ atrogin-1 /MuRF1 signalling pathway plays a crucial role in skeletal muscle atrophy of COPD.