Objective To investigate the effect of Ligustrazine on the expressions of FoXO3a, MAFbx, and MuRF1 indenervated skeletal muscle atrophy rats. Methods Fifty-four 8-week-old female Sprague Dawley rats were randomly dividedinto 3 groups: normal control group (group A, n=6), denervated control group (group B, n=24), and Ligustrazine interventiongroup (group C, n=24). After the denervated gastrocnemius models were established in the rats of groups B and C, sal ine andLigustrazine [80 mg/(kg·d)] were given every day by intraperitoneal injection, respectively. However, no treatment was donein group A. At 2, 7, 14, and 28 days after denervation, the wet weight of gastrocnemius was measured to calculate the ratio ofwet weight. The mRNA and protein expression levels of FoXO3a, MAFbx, and MuRF1 were detected by RT-PCR and Westernblot. Results The ratio of gastrocnemius wet weight decreased with time after denervation in groups B and C, showingsignificant differences when compared with that of group A (P lt; 0.05), and group C were significantly higher than that of groupB at 7, 14, and 28 days (P lt; 0.05). The mRNA and protein expressions of FoXO3a, MAFbx, and MuRF1 in groups B and Cwere significantly higher than those in group C at 7, 14, and 28 days (P lt; 0.05), and group C was significantly lower than groupB (P lt; 0.05). Conclusion Ligustrazine may postpone denervated skeletal muscle atrophy by reducing mRNA and proteinexpressions of FoXO3a, MAFbx, and MuRF1.
Objective To analyze the expression changes of Pax7 and MyoD in satell ite cells of denervated skeletal muscle at the mRNA and protein level, and to explore the pathway concerning with denervation and regeneration of skeletal muscle. Methods Nine male SD rats of 2 months old, weighing 180-200 g were selected. The right gastrocnemius musclewas made denervation model as experimental sides, and the same operation was performed in the left sides as control. The immunofluorescence analysis was done for detecting the protein expressions of Pax7 and MyoD in muscles denervated 3, 7 and 21 days after operation. The RT-PCR analysis was used to explore the mRNA expression of MyoD and Pax7 in extracted tissues from denervated muscles. Results Nine rats survived after operation, with l imited motion of right hind l imbs, and normal motion of left l imbs. The result of immunofluorescence with the polyclonal antibody against MyoD and the monoclonal antibody against Pax7 showed that the number of MyoD positive cells in prolonged denervated muscles (experimental sides) increased gradually, and reached the peak 21 days after denervation, there was significant difference compared with that of control sides (P lt; 0.05). However, the total number of Pax7 positive cells decreased with the prolongation of denervation, the expression of Pax7 was b only at 3 days, then decreased gradually, and there was no positive cells in denervated muscles of 21 days. There were significant differences in Pax7 positive cells between experimental sides and control sides at 3 and 7 days (P lt; 0.05). The results of immunostaining showed that MyoD mRNA levels increased , and reached the peak at 21 days of denervation, there was significant difference compared with that of control sides (P lt; 0.05). Whereas, Pax7 mRNA expressed only in control, denervated muscles of 3 and 7 days, and its expression level decreased gradually. Conclusion One possible reason of the depletion of satell ite cell pool in prolonged denervated muscle is the down-regulation of Pax7.
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 effect of the neuromuscular pedicle transplantation in prevention against atrophy in the denervated muscle. Methods Fortyeight SD rats were used to establish the right side tibialis anterior muscle denervation model. The long peroneal muscle neuromuscular pedicle was made as a treatment in 12 rats (Group A); the nerve shaft embedding was used in 12 rats (Group B); no treatment was used in 12 rats(Group C); the remaining 12 rats were used as normal controls (Group D). The gait analysis, electromyogram，muscle wet weight, and muscle fiber crosssectional area were used to determine and compare the effect of the operation at 6 and 12 weeks postoperatively. ResultsAt 6 weeks postoperatively, the parameters tested in Group A about the gait analysis (peroneal function index, PFI, －47.20±12.30), electromyogram, muscle wet weight (0.384 0±0.024 6 g)and muscle fiber cross-sectional area (1 040.98±120.54 μm2) were significantly better than those in Group C (PFI, -114.40±14.84; muscle wet weight, 0.173 0±0.019 1 g; muscle fiber cross-sectional area, 585.08±182.93 μm2,Plt;0.05), and the final two parameters were significantly better than those in Group B (0.294 0±0.056 4 g,763.92±82.68 μm2,Plt;0.05). At 12 weeks postoperatively, the musclefiber crosssectional area in Group A(1 360.10±261.45 μm2) had no significant difference from that in Group D (1 544.57±266.92 μm2，Pgt;0.05)，and most of the parameters tested in Group A were better than those in Groups B and C. Conclusion Neuromuscular pedicle transplantation has an excellent effect in prevention against atrophy in the denervated muscle, and the effect of neuromuscular pedicle transplantation is better than that of the nerve shaft embedding.
Objective To explore the rule of changes in the myoblast stem cells (satellite cells) in the denervated and innervated muscles and to find out thecellular mechanism of the changes in the muscle morphology and function. Methods The denervated muscleatrophy models were established from 27 Wistar rats aged 1 month. One to six months after operation, examinations of the histology, histochemistry, and morphology were performed on the specimens from the bilateral triceps muscle of the calves of 3 rats in each month. Meanwhile, examination of the cell biology was performed on the specimens from the bilateral triceps muscleof the calves of 1 rat 1, 2 and 3 weeks after operation, and monthly for 1-6 months after operation. The innervation models were established from 35 Wistar rats aged 1 month. Immediately after the denervation, and monthly for 1-6 months after operation, 5 denervated rats underwent the nerve implantation. The changes in the electrophysiological index were observed dynamically until 8 weeks after the nerve implantation.Results After the denervation, the muscle wet weight and the muscle cell area decreased rapidly, but the content of the collagen fibers increased gradually. The number of the nucleus in the period of proliferationwas the greatest 3-4 months after the denervation, and then decreased rapidly.The muscle satellite cells began to increase obviously 3 weeks after the denervation, but 2 months later they decreased rapidly and 4 months later the number of the cells was the smallest. Four to five weeks after innervation, the muscle action potential could be induced, and the best innervation effect could be achieved in the implanted nerve after the 2-3 months on denervation, and at this time the differentiation ability of the satellite cells was the best. Conclusion Four months after the denervation of the skeletal muscle, an extremely small number of the satellite cells can make the muscle enter the irreversible atrophy. However, when the innervation is performed 2-3 months after the denervation, the actively-growing satellite cells can promote a better functional recovery ofthe atrophic muscle.
Objective To study the effect of the competitive inhibitor of nitric oxide synthase NG-nitro-L-arginine methyl ester (LNAME) on thedenervated muscle atrophy. Methods A model of the denervated gastrocnemius atthe right lower limb was established in 36 SD adult rats. The rats were randomly divided into two groups: the L-NAMEgroup (Group A) and the control group(Group B). L-NAME 10 mg/ kg daily was injected into the denervated gastrocnemius inGroup A, and normal saline was injected into the denervated gastrocnemius in Group B. At 2, 4 and 8 weeks after operation, the rate of the muscle wet weight preservation, the cross section area of the myocyte, the protein amount, and the percentage of the apoptotic muscle cells were measured respectively and the ultramicrostructure of the myocyte was observed. Results At 2 and 4 weeks after operation, the rate of the muscle wet weight preservation, the cross section area of themyocyte, and the protein amount were significantly greater in Group A than in Group B; however, the percentage of the apoptotic muscle cells was significantly smaller in Group A than in Group B. The observation of the ultramicrostructure of themyocyte showed that an injection of L-NAME could protect the ultramicrostructure of themyocyte. At 8 weeks after operation, there was no significant difference between the two groups in the abovementioned parameters. Conclusion The nitric oxide synthase inhibition can delay the denervated muscle atrophy.
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.
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 clarify the character of nerve growth factor (NGF) in the denervated red and white muscles and the relationship between the amount of NGF and sensitive neurotrophism of denervated red and white muscles. METHODS: The model of the denervated gastrocnemius and soleus was made by clipping the sciatic nerve of Wister rats. The immunohistochemistry was taken to measure the amount of NGF in muscles, and the neurotrophism of extracts of muscles was tested with culture of dorsal root ganglions at the 1st, 3rd, 7th and 14th days after injury. RESULTS: The amount of NGF in denervated gastrocnemius and soleus decreased, especially in soleus. The neruotrophism of the extracts of the two kinds of denervated muscles did not decrease; on the contrary, it increased after a week after injury. CONCLUSION: The injury of peripheral nerves causes the amount of NGF in the target tissues to increase, but the change is different between the denervated muscles; the neurotrophism of the extracts of musclesis determined by all kinds of neurotrophic factors, and can not be explained by a single factor.
OBJECTIVE To observe the degeneration and regeneration of the Meissner’s corpuscles after implanted sensory nerve into the denervated monkey’s fingers under electron microscope. METHODS The two finger nerves of the monkey’s fingers were denervated. Afterwards, one finger nerve was cut off, and the other was reimplanted into the denervated finger. After 1, 3, 5, 8 and 12 months, the finger skin was cut off and observed under electron microscope. RESULTS The degenerative changes of nerve ending in Meissner’s corpuscles were observed after 1 month of denervation, and the basic structure of the corpuscles had no obvious changes. After 3 months, the axons of corpuscles were disappeared, and the volume of corpuscles was shrunk. The basic structure of nerves was disappeared, and the lemmocyte and neurolemma plate were changed after 5 months. The collagen fibrils in the corpuscles were gradually increased in 8 months, the endoneurial structure and interneurial matrix were completely disappeared and replaced by collagen fibrils in 12 months. After 3 months of nerve implantation, unmyelinated nerve fibers were appeared and grew into the corpuscles. A part of corpuscles innervated in 5 months. Most of corpuscles innervated and myelinated nerve fibers were observed in 8 months. And in 12 months, corpuscles innervated to normal level. CONCLUSION The implantative sensory nerve by means of reinnervating the original corpuscles and regenerating new corpuscles could innervate the degenerative Meissner’s corpuscles.