经皮电刺激大鼠骨骼肌失神经肌萎缩时成肌调节因子基因的表达

背景：在去神经早期大鼠骨骼肌成肌调节因子(MyoD)表达明显上调,有明显延缓骨骼肌肌萎缩的作用。临床实验证实电刺激是治疗失神经肌萎缩的有效方法。尚未有实验证实电刺激对失神经肌萎缩MyoD表达的影响。 目的：验证电刺激对大鼠骨骼肌MyoD基因表达的影响。 设计、时间及地点：随机对照动物实验,于2008-07/11在山西医科大学动物实验中心完成。 材料：健康的SD大鼠36只,雌雄不限。随机分成3组,即空白对照组、去神经组、电刺激组,每组12只。 方法：空白对照组不做任何处理;去神经组和电刺激组大鼠制作右侧坐骨神经离断,腓肠肌失神经支配模型。用电刺激对电刺激组进行刺激,1次/d,30 min/次。分别于去神经第2,7,14,28天,处死大鼠,取小腿的腓肠肌肉标本。 主要观察指标：用反转录聚合酶链式反应技术检测MyoD mRNA的表达变化,免疫组织化学检测MyoD蛋白表达的变化。 结果：在去神经支配后第2,7,14,28天,去神经组和电刺激组标本中MyoDmRNA和蛋白含量表达上调,与空白对照组比较差异有显著性意义(P < 0.05),电刺激组表达高于去神经组(P < 0.05)。 结论：通过电刺激可以上调大鼠腓肠肌失神经模型MyoD的表达,说明电刺激是延缓骨骼失神经肌萎缩的有效方法。     

Activity-unrelated neural control of myogenic factors in a slow muscle

The properties of skeletal muscles are modulated by neural and nonneural factors, and the neural factors can be modulated by activity-independent as well as activity-dependent mechanisms. Given that daily activation of fast muscles is considerably less than of slow muscles, we hypothesized that the myogenic properties of the rat soleus (a slow muscle) would be more dependent on activity-dependent than activity-independent factors. Muscle mass, MyoD, and myogenin mRNA and protein levels, and satellite cell proliferation and differentiation rates (bromodeoxyuridine incorporation) were examined at 3, 14, and 28 days after either spinal cord isolation (SI, neuromuscular connectivity intact with minimal activation) or denervation (no neural influence). Soleus atrophy was similar in the SI and denervated groups at each time point, although increases in whole-muscle expression of myogenin and, to a lesser degree, MyoD were lower (P < 0.05) in SI than denervated soleus muscles. Proliferation and differentiation of satellite cells, as well as mitotic activity of connective tissue cells, were lower (P < 0.05) in SI than denervated soleus muscles. In some instances, these changes were not observed until the later time points, i.e., 14 or 28 days. These results demonstrate that the motoneurons that innervate the slow soleus muscle have a significant modulatory influence on some muscle properties via mechanisms that are independent of activation. These activity-independent modulatory influences, however, are less in the slow soleus than previously observed in fast muscles.     

成肌调节因子Myf-5在失神经骨骼肌萎缩不同时段的表达

背景：成肌调节因子Myf-5是参与肌肉发生过程分子调控、启动和维持骨骼肌细胞生长发育的重要基因,可能与失神经骨骼肌萎缩的发生有关。 目的：观察不同部位、不同时段骨骼肌失神经支配后成肌调节因子Myf-5基因的表达情况。 设计、时间及地点：随机对照动物实验,于2008-03/04在山西医科大学完成。 材料：选择健康8周龄雄性SD大鼠24只,随机分成4组,即假手术组(有神经支配)、去神经2 d组、去神经7 d组、去神经28 d组,每组6只。 方法：假手术组不切断坐骨神经,仅做假手术。去神经组右下肢后部中段切断坐骨神经1 cm以上,分别于去神经第2,7,28天用脊椎脱臼法处死大鼠,分离出右小腿的胫骨前肌、比目鱼肌、腓肠肌、跖肌标本。 主要观察指标：用反转录-聚合酶链反应技术检测各组肌肉Myf5 mRNA表达情况,抗Myf-5 多克隆抗体免疫组织化学染色(ABC 法),测量灰度值。 结果：去神经骨骼肌早期,Myf-5的mRNA在去神经支配后第2,7,28天均表达上调(P < 0.05)。Myf-5 抗体阳性染色细胞核数在SD大鼠骨骼肌失神经28 d时的肌卫星细胞中最多。 结论：Myf-5在大鼠失神经骨骼肌萎缩早期不同肌肉表达均为上调。大鼠骨骼肌失神经支配后早期肌卫星细胞中Myf-5表达上调。     

Bcl-2 and bax immunohistochemistry in denervation-reinnervation and necrosis-regeneration of rat skeletal muscles

Bcl-2 and Bax immunohistochemistry was examined in the skeletal muscle of rats after cutting the sciatic nerve, as a model of denervation and reinnervation, and in the anterior tibialis muscle of rats after an intramuscular injection of metoclopramide, as a model of necrosis and regeneration of muscle fibers, to better understand the role of these proteins in muscle disorders. An increase in Bax immunoreactivity was seen in long-standing denervated and reinnervated muscle fibers. Bcl-2 and Bax immunoreactivity was limited to macrophages in necrotic muscle fibers at 24 h after the intramuscular injection of metoclopramide. However, increased Bax immunoreactivity was observed in regenerating muscle fibers by the fourth day after the injection. Muscle fiber nuclei with the morphological features of apoptosis were not observed in rat muscles after the intramuscular injection of metoclopramide or after the severing of the sciatic nerve. Furthermore, using the Tunel method, no stained nuclei were observed in the two groups of animals. Our observations in the experimental models of skeletal muscle denervation-reinnervation and necrosis-regeneration here described suggest that modification in the intensity of the Bax protein is not related to the process of cell death but rather that increased Bax expression is associated with muscle fiber regeneration.     

PAX3/7 expression coincides with MyoD during chronic skeletal muscle overload

Paired box (Pax) proteins 3 and 7 are key determinants for embryonic skeletal muscle development by initiating myogenic regulatory factor (MRF) gene expression. We show that Pax3 and 7 participate in adult skeletal muscle plasticity during the initial responses to chronic overload (< or =7 days) and appear to coordinate MyoD expression, a member of the MRF family of genes. Pax3 and 7 mRNA were higher than control within 12 h after initiation of overload, preceded the increase in MyoD mRNA on day 1, and peaked on day 2. On days 3 and 7, Pax7 mRNA remained higher than control, suggesting that satellite cell self-renewal was occurring. Pax3 and 7 and MyoD protein levels were higher than control on days 2 and 3. These data indicate that Pax3 and 7 coordinate the recapitulation of developmental-like regulatory mechanisms in response to growth-inducing stimuli in adult skeletal muscle, presumably through activation of satellite cells.     

Polyamine accumulation in normal and denervated neonatal muscle

Polyamines were monitored in normal and denervated neonatal rat gastrocnemius muscle as markers of the degeneration-regeneration processes in muscle characteristic of progressive neuromuscular disorders. The muscle was denervated within 6 h of birth. The highest polyamine concentrations were present at day 2 of birth and remained elevated during the first week, the period of most rapid muscle growth. Denervation did not alter the concentration of any of the polyamines compared with normal or contralateral controls during the first 3 weeks postparturition. These data suggest that neonatal polyamine synthesis, an index of extent of cell growth processes, is not affected by denervation. However, prolonged denervation did alter the polyamine accumulation patterns, with denervated muscle having significantly elevated spermine at 21 days, putrescine and spermidine concentrations at 28 days, and elevated spermidine at 56 days of age. Within 56 days, denervated muscle compared with normal innervated contralateral control muscle contained fourfold and twofold more spermidine and spermine per milligram protein. In conclusion, it is likely that the continued elevation of polyamines in denervated muscle represents the conservation of a metabolic pattern characteristic of immature muscle, as it is known that neonatal denervation results in failure of muscle maturation and differentiation. However, fibroblast proliferation also may be a contributing factor to the markedly altered polyamine concentrations found in denervated muscle.     

Fibrillation potentials of denervated rat skeletal muscle are associated with expression of cardiac-type voltage-gated sodium channel isoform Nav1.5

ObjectiveThe molecular mechanisms underlying fibrillation potentials are still unclear. We hypothesised that expression of the cardiac-type voltage-gated sodium channel isoform Nav1.5 in denervated rat skeletal muscle is associated with the generation of such potentials.MethodsMuscle samples were extracted and analysed biologically from surgically denervated rat extensor digitorum longus muscle after concentric needle electromyographic recording at various time points after denervation (4 h to 6 days).ResultsBoth nav1.5 messenger RNA (mRNA) signal on northern blotting and Nav1.5 protein expression on immunohistochemistry appeared on the second day after denervation, exactly when fibrillation potentials appeared. Administration of lidocaine, which has much stronger affinity for sodium channels in cardiac muscle than for those in skeletal muscle, dramatically decreased fibrillation potentials, but had no effect on contralateral compound muscle action potentials.ConclusionsExpression of Nav1.5 participates in the generation of fibrillation potentials in denervated rat skeletal muscle.SignificanceWe proposed an altered expression of voltage-gated sodium channel isoforms as a novel mechanism to explain the occurrence of fibrillation potentials following skeletal muscle denervation.     

失神经骨骼肌细胞凋亡：运动与感觉神经的比较

Skeletal muscle atrophy inevitably occurs in denervated skeletal muscle,and cell apoptosis plays an important role in skeletal muscle atrophy and degeneration.The present study established rat models of simple nerve injury by transecting the ventral or dorsal spinal nerve root and observed rat skeletal muscle cell apoptosis following simple motor nerve injury versus simple sensory nerve injury.Following skeletal muscle denervation for 10 weeks,cell apoptosis was detected in skeletal muscle,which was accompanied by obvious changes in rat behavior and electrophysiological responses.In addition,changes in cross-sectional area and average gray-scale of motor endplates of the gastrocnemius muscle were analyzed following sciatic nerve injury and motor nerve injury.Cell nuclei in denervated skeletal muscle tissue were more densely arranged than in normal skeletal muscle tissue.Cell nuclei were most dense in the sciatic nerve injury group,followed by the motor nerve injury group and the sensory nerve injury group.Fas/FasL expression and the number of apoptotic cells increased in denervated skeletal muscle,and apoptosis-related changes were observed.These findings suggested that motor and sensory nerves provided trophic actions following skeletal muscle and motor nerve injury,resulting in a greater influence on skeletal muscle atrophy than sensory nerve injury.Therefore,reconstruction of motor nerves should be preferentially considered for treating denervation-induced skeletal muscle atrophy.     

Role of MyoD in denervated, disused, and exercised muscle

The myogenic regulatory factor MyoD plays an important role in embryonic and adult skeletal muscle growth. Even though it is best known as a marker for activated satellite cells, it is also expressed in myonuclei, and its expression can be induced by a variety of different conditions. Several model systems have been used to study the mechanisms behind MyoD regulation, such as exercise, stretch, disuse, and denervation. Since MyoD reacts in a highly muscle-specific manner, and its expression varies over time and between species, universally valid predictions and explanations for changes in MyoD expression are not possible. This review explores the complex role of MyoD in muscle plasticity by evaluating the induction of MyoD expression in the context of muscle composition and electrical and mechanical stimulation.     

Fos immunoreactivity in the diagonal band and the perinuclear zone of the supraoptic nucleus after hypertension and hypervolaemia in unanaesthetized rats

We used Fos immunocytochemistry to study the effects of hypertension and hypervolaemia on neurones in the diagonal band of Broca and the perinuclear zone of the supraoptic nucleus, two nuclei that are both involved in the baroreceptor regulation of vasopressin neurones in the supraoptic nucleus. In addition, we used sino-aortic denervation to examine the role of arterial baroreceptors in the response to these haemodynamic changes. Sham-operated and sino-aortic denervated rats were infused with phenylephrine sufficient to increase blood pressure for 2 h. Control rats were infused with the same volume of isontonic saline. Only Sham sino-aortic denervated rats showed reflex bradycardia in response to the increased blood pressure. Volume expansion was produced by infusing the rats with isotonic saline equal to 10% of their body weight for 10 min, which significantly increased central venous pressure. In the diagonal band of Broca and the perinuclear zone, the number of Fos-positive neurones was significantly increased after phenylephrine infusion. Sino-aortic denervation blocked the significant increase in both regions. After volume expansion, a significant increase in Fos staining was observed only in the perinuclear zone of the supraoptic nucleus. This increase was not blocked by sino-aortic denervation. Our results indicate that both the diagonal band of Broca and the perinuclear zone of the supraoptic nucleus are activated by stimulating arterial baroreceptors; however, the perinuclear zone of the supraoptic nucleus is stimulated during volume expansion. Furthermore, the activation of perinuclear zone of the supraoptic nucleus after volume expansion is not dependent on intact arterial baroreceptors.     

Parvalbumin immunohistochemistry in denervated skeletal muscle

Parvalbumin is a calcium–binding protein which, in muscle, is mainly found in type 2B fibres, whereas type 1 fibres lack parvalbumin immunoreactivity. Previous studies have shown that this pattern is highly dependent upon motor neuron innervation and is modified in denervated, cross–reinnervated or chronic low–frequency stimulated muscles. In the present study, we have examined the modifications of parvalbumin immunocyto–chemistry in the anterior tibialis muscle of the rat at different intervals following section of the sciatic nerve. During the first 2 weeks after denervation, no changes in parvalbumin immunoreactivity were seen, although a global reduction of fibre diameter was observed. Three weeks after denervation, small angulated. strongly parvalbumin–immunoreactive fibres appeared. From the second month onwards, the pattern of parvalbumin immunohistochemistry was characterized by areas composed of small, strongly immunoreactive fibres separated by less atrophic areas displaying a normal chequerboard distribution of parvalbumin immunoreactivity. The increase of parvalbumin–immunoreactivity in denervated and reinnervated muscle, as seen in our study, indicates that important changes in parvalbumin distribution occurs in muscle fibres after denervation. These changes are probably produced in an attempt to bind the free cytosolic calcium which accumulates in denervated fibres, and further reinforces the role of parvalbumin in calcium homeostasis during denervation and reinnervation.