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 explore the effect of tri pterygium glycoside (TG) on the skeletal muscle atrophy and apoptosis after nerve allograft. Methods Twenty Wistar male rats were adopted as donors, weighing 200-250 g, and the sciatic nerves were harvested. Fifty SD male rats were adopted as recipients, weighing 200-250 g. Fifty SD rats were made the models of10 mm right sciatic nerve defect randomly divided into five groups (n=10): group A, group B, group C, group D and group E.groups A and B received fresh nerve allograft, groups C and D received sciatic nerve allograft pretreated with TG, and group E received autograft. The SD rats were given medicine for 5 weeks from the second day after the transplantation: groups A and E were given physiological sal ine, groups B and D TG 5 mg/ (kg·d), and group C TG 2.5 mg/ (kg·d). At 3 and 6 weeks, respectively, after nerve transplantation, general observation was performed; the structure of skeletal muscles was observed by HE staining; the diameter of skeletal muscles was analyzed with Image-Pro Plus v5.2; the ultrastructure of skeletal muscles was observed by TEM; the expressions of Bax and Bcl-2 were detected by immunohistochemical staining; and the apoptosis of skeletal muscles was detected by TUNEL. Results All rats survived to the end of the experiment. In general observation, the skeletal muscles of SD rates atrophied to different degrees 3 weeks after operation. The muscular atrophy in group A was more serious at 6 weeks, and that in the other groups improved. The wet weight, fiber diameter and expression of Bcl-2 in group A were significantly lower than those in groups B, C, D and E (P lt; 0.01)；those in groups B, C and D were lower than those in group E (P lt; 0.05); and there were no significant differences among groups B, C and D (P gt; 0.05). The apoptosis index and expression of Bax in group A were significantly higher than those in groups B, C, D and E (P lt; 0.01)；those in groups B, C and D were higher than in groupE (Plt; 0.05); and there were no significant differences among groups B, C and D (P gt; 0.05). Three weeks after nerve allograft, under the l ight microscope, the muscle fibers became thin; under the TEM, the sarcoplasmic reticulum was expanded. Six weeks after nerve allograft, under the l ight microscope, the gap of the muscle fibers in group A was found to broaden and connective tissue hyperplasia occurred obviously; under the TEM, sarcomere damage, serious silk dissolution and fragmentary Z l ines were seen in group A, but the myofibrils were arranged tidily in the other groups, and the l ight band, dark band and sarcomere were clear. Conclusion TG can decrease the skeletal muscle atrophy and apoptosis after nerve allograft. The donor’s nerve that is pretreated with TG can reduce the dosage of immunosuppressant for the recipient after allograft.
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.
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 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 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 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 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 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.