Objective To investigate the early change of brain-derived neurotrophic factor (BDNF) in denervated red and white muscles and the regeneration of nerves innervating the muscles and to discuss the effect of the target organs on regeneration of the injured nerves.Methods Forty Wistar rats were divided into 5 groups. The sciatic nerves in 4 groups were sheared to make the models of the denervated muscles and the other one as control group. The amount of BDNF in muscles was measured with immunohistochemistry 1 day, 3 days, 7 days and 14 days after injury. The models of the regeneration of the nerves were made in another 15 rats whose sciatic nerves were disconnected with forceps. The nerve conduction velocity and electromyogram were tested with neuroelectrophysiology7 days and 14 days after injury. Results The expression of BDNF in soleus increased significantly on the 1st day, the 3rd day and the 7th day (P＜0.01); theexpression ingastrocnemius was lower, but there was no significant difference(P＞0.05) on the 1st day, the 3rd day,the 7th day and the 14th day when compared with control group. After 14 days of injury in the nerves innervating GAS and SOL, the nerve conduction velocities and the amplitudes of wave M recovered to (36.60±7.40)% and (19.9±6.4)% of normal value, and (42.50±3.50)% and (13.7±4.0)% of normal value respectively; there were no significant differences between the two muscles(P＞0.05).Conclusion There is- difference in BDNF amount between the denervated red and white muscles, but the recovery of the two kinds of the motornerves is similar,and the neurotrophism of denervated muscles was determined by all kinds of neurotrophic factors.
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.
OBJECTIVE: To define how to preserve the severed limbs to prolong the period of replantation. METHODS: The original articles about preservation of severed limbs in recent years were reviewed, it was suggested that the period of replantation was determined by the injury of skeletal muscle. RESULTS: When the environment of severed limbs was changed, the injures of skeletal muscle could be decreased. CONCLUSION: After the severed limbs are reasonably preserved, the period of replantation may be prolonged.
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.
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.
【Abstract】 Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not. Methods Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (MbGDNF). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n=27) or MbGDNF-scaffold complex (MbGDNF group, n=27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used. Results MbGDNF could highly expressed GDNF gene. Mb and MbGDNF could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of actylcholine receptor α1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 ± 6.6, 227.3 ± 8.5, and 173.3 ± 9.1, respectively in MbGDNF group, and were 234.7 ± 5.5, 196.0 ± 13.5, and 166.7 ± 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P lt; 0.05), no significant difference at 12 weeks (P gt; 0.05). Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
OBJECTIVE: To observe the changes of heme oxygenase-1 (HO-1) expression in the skeletal muscle after ischemia-reperfusion of hind limb in rats. METHODS: A model of hind limb ischemia was made by clamping femoral artery with a microvascular clip. Soleus muscle was obtained from the animals received sham operation, 4 h ischemia without reperfusion and 2 h, 4 h, 8 h, 16 h, 24 h reperfusion after 4 h ischemia. Soleus histology and malondialdehyde (MDA) content were measured. The levels of HO-1 mRNA and protein were measured in different time by Northern blotting, Western blotting and immunohistochemistry technique. RESULTS: After ischemia-reperfusion of limb, HO-1 mRNA increased at the 2nd hour, reached a peak at the 8th hour, and returned toward baseline at the 24th hour. The change of protein level was essentially in agreement with that of mRNA. Immunohistochemical results showed that HO-1 expressed primarily in skeletal muscle cytoplasma. There were no positive signals of mRNA and protein in sham group and in ischemia group. After limb reperfusion, MDA contents in the soleus muscle increased significantly when compared with that in the sham group (P lt; 0.05). MDA content of the 8th after reperfusion decreased significantly when compared with that of the 4 h after reperfusion (P lt; 0.05). CONCLUSION: Ischemia-reperfusion can induce HO-1 expression in skeletal muscle in rats, which may provide protection for injured tissue.
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.
Skeletal muscle and metabolic function are important factors affecting the health status of the elderly. Branched-chain amino acids (BCAA) can improve muscle recovery, reduce muscle soreness after exercise, and BCAA can also enhance metabolic health, helping to regulate blood sugar levels and improve insulin sensitivity in the elderly. In addition, BCAA can improve cognitive function, reducing the risk of age-related cognitive decline. This article reviews the relationship between BCAA and aging, skeletal muscle, and metabolic diseases, explaining how BCAA can support and promote muscle mass and function in the elderly, as well as have a positive impact on metabolic health and cognitive function.