In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding β‐galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.     

活体电穿孔方法提高外源GRF基因在小鼠肌肉组织中的表达

向小鼠后肢小腿肌注pGRF表达质粒并加以电穿孔条件,注射剂量分别为5,10,50μg,于注射前及注射后10,20,30d测体质量,并采血分离血清测血清GRF水平。结果显示,5μgpGRF质粒电穿孔组注射后10dGRF分别比对照组、5μgpGRF质粒组升高44.73%(P0.05),12.86%(P0.05);血清中GRF水平升高。30d累积增重,5μgpGRF质粒电穿孔组分别比对照组、5μgpGRF质粒组高11.46%,6.75%(P0.05);10μgpGRF质粒组分别比对照组和10μgpGRF质粒电穿孔组高19.52%(P0.05),19.42%(P0.05)。50μgpGRF质粒组45d累积增重分别比对照组、pGRF质粒电穿孔组高14.00%,12.00%(P0.05)。结果表明,电穿孔处理可提高GRF基因在小鼠肌肉中的表达。     

Ex vivo bupivacaine treatment results in increased adipogenesis of skeletal muscle cells in the rat

Intramuscular adipose tissue (IMAT) is observed in some skeletal muscle pathologies. IMAT is implicated not only in the disorders of muscle contraction, but also of metabolism and insulin sensitivity due to its nature as a secretary organ. Several studies indicate the presence of cells with adipogenic potential in skeletal muscle. However, the mechanism of fate specification that triggers these cells to enter an adipogenic program in vivo remains to be solved. In the present study, we examined whether activation of the adipogenic program of muscle‐resident cells precedes their proliferation upon muscle injury. For this purpose, muscle injury was induced by injecting bupivacaine (BPVC) to excised skeletal muscle ex vivo. Cells isolated from ex vivo BPVC‐treated muscle exhibited higher adipogenic potential than those from saline‐treated muscle. Pre‐plating exposure of skeletal muscle cells to basic fibroblast growth factor (bFGF) mimicked the effect of ex vivo BPVC‐treatment, suggesting that bFGF released from extracellular matrix in response to muscle injury activates their adipogenic program. Interestingly, the number of myotubes were significantly reduced in the culture from BPVC‐treated muscle, suggesting that adipocytes negatively regulate myogenesis.     

Suppression of MyoD induces spontaneous adipogenesis in skeletal muscle progenitor cell culture

The degree of intramuscular adipose tissue accumulation is one of the factors affecting meat quality. Accumulation of adipocytes is also observed under the pathological condition of skeletal muscle such as muscular dystrophy and sarcopenia. The origin of adipocytes seen in skeletal muscle is mesenchymal progenitor cells that can give rise to both adipocytes and fibroblasts. In the present study, we demonstrated that siRNA-mediated suppression of MyoD expression in rat skeletal muscle progenitor cell culture, which comprises both myogenic satellite cells and mesenchymal progenitor cells, resulted in diminished myotube formation and an unexpected spontaneous appearance of white adipocytes. Suppressing myomaker expression also resulted in complete absence of myotube formation without reducing MyoD expression, but no adipogenesis was seen in this scenario, indicating that decline in MyoD expression rather than decreased myotube formation is necessary to induce adipogenesis. In addition, spontaneous adipogenesis induced by suppressing MyoD expression in culture was inhibited by the conditioned medium from control culture, indicating that anti-adipogenic factor(s) are secreted from MyoD-positive myogenic cells. These results indicate the presence of regulatory mechanism on adipogenesis by myogenic cells.     

Technique for physiological examination of canine skeletal muscle in vivo

A technique is described for studying the physiological function of canine skeletal muscle in vivo. The contractile properties of the tarsal flexor muscles were examined in three beagle dogs under general anaesthesia. The force responses to electrical stimulation of the common peroneal nerve were measured at various frequencies to determine the frequency:force relationship for this muscle group. Fatigue characteristics were also examined during intermittent stimulated activity delivered in a set pattern of frequencies. The results provide quantitative characterisation of muscle function which is repeatable. The technique described could be applied to other animals and is a potentially powerful tool for evaluating the effects of drugs on muscle performance.     

Differentiation of skeletal muscle Mesenchymal progenitor cells to myofibroblasts is reversible

Accumulation of intramuscular adipose tissue (IMAT) and development of fibrous tissues due to accumulation of collagen both affect meat quality such as tenderness, texture, and flavor. Thus, it is important for the production of high‐quality meat to regulate the amount of adipose and fibrous tissues in skeletal muscle. IMAT is comprised of adipocytes, while collagens included in fibrous tissues are mainly produced by activated fibroblasts. Both adipocytes and fibroblasts are differentiated from their common ancestors, called mesenchymal progenitor cells (MPC). We previously established rat MPC clone, 2G11 cells. As several reports implicated the plasticity of fibroblast differentiation, in the present study, using 2G11 cells, we asked whether myofibroblasts differentiated from MPC are capable of re‐gaining adipogenic potential in vitro. By treating with bFGF, their αSMA expression was reduced and adipogenic potential was restored partially. Furthermore, by lowering cell density together with bFGF treatment, 2G11 cell‐derived myofibroblasts lost αSMA expression and showed the highest adipogenic potential, and this was along with their morphological change from flattened‐ to spindle‐like shape, which is typically observed with MPC. These results indicated that MPC‐derived myofibroblasts could re‐acquire adipogenic potential, possibly mediated through returning to an undifferentiated MPC‐like state.     

AMP-activated protein kinase and adipogenesis in sheep fetal skeletal muscle and 3T3-L1 cells

Marbling, or i.m. fat, is an important factor determining beef quality. Both adipogenesis and hypertrophy of existing adipocytes contribute to enhanced marbling. We hypothesized that the fetal stage is important for the formation of i.m. adipocytes and that AMP-activated protein kinase (AMPK) has a key role in adipogenesis during this stage. The objective of this study was to assess the role of AMPK in adipogenesis in fetal sheep muscle and 3T3-L1 cells. Nonpregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n = 7) or overfed (OF, 150% of NRC, n = 7) diet from 60 d before to 75 d after conception, when the ewes were killed. The fetal LM was collected at necropsy for biochemical analyses. The activity of AMPK was less in the fetal muscle of OF sheep. The expression of peroxisome proliferator-activated receptor (PPAR)gamma, a marker of adipogenesis, was greater in OF fetal muscle compared with Con fetal muscle. To further show the role of AMPK in adipogenesis, we used 3T3-L1 cells. The 3T3-L1 cells were incubated in a standard adipogenic medium for 24 h and 10 d. Activation of AMPK by 5-aminoimidazole-4-car-boxamide-1-beta-d-ribonucleoside dramatically inhibited the expression of PPARgamma and reduced the presence of adipocytes after 10 d of differentiation. Inhibition of AMPK by compound C enhanced the expression of PPARgamma. In conclusion, these data show that AMPK activity is inversely related to adipogenesis in fetal sheep muscle and 3T3-L1 cells.     

Preliminary experiments on mechanical stretch-induced activation of skeletal muscle satellite cells in vivo

We have shown in vitro that mechanical stretch triggers activation of quiescent satellite cells of skeletal muscle to enter the cell cycle through an intracellular cascade of events including nitric oxide (NO) synthesis that results in the release of hepatocyte growth factor (HGF) from its extracellular association and its subsequent presentation to signaling receptors. In order to explore the activation mechanism in vivo, stretch experiments were conducted in the living animal using our suspension model developed. This system used the weight of the hind portion of rats to stretch the inside muscles of the left hind limb suspended for a period of 0.5–2.0 h. At the end of the stretch period, the rats received an intraperitoneal injection of bromodeoxyuridine followed by immunocytochemistry for its incorporation as an index of satellite cell activation in vivo. Depending on the period of stretch, bromodeoxyuridine labeling was increased significantly over the contralateral unstretched leg or control muscle from untreated rats. A stretched muscle extract prepared from the 2 h stretched tissue by incubating it in PBS, showed the active form of HGF as revealed by immunoblotting and it could stimulate the activation of unstretched satellite cells. Also, administering NO synthase inhibitor L‐NAME prior to muscle stretch abolished the stretch activation of satellite cells. Therefore, the results from these experiments demonstrate that stretching muscle triggers NO synthesis and HGF release, which could activate satellite cells in vivo.     

In vitro characteristics of normal and dystrophic skeletal muscle from dogs

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (post-fusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.     

Sensitivity of skeletal muscle to pro-apoptotic factors

In mononuclear cells, apoptosis leads to DNA fragmentation and cell destruction, regardless of the activated pathway. As regards multinuclear cells, e.g. skeletal muscle fibers, apoptosis rarely induces the death of the entire cell, and it generally affects single nuclei. This process, referred to as nuclear apoptosis, has a negative effect on the expression of genes in the myonuclear domain. Apoptosis may be initiated in muscle cells by external stimuli which activate cell membrane death receptors as well as by internal stimuli which stimulate the mitochondrial release of pro-apoptotic proteins. Reactive oxygen species also play an important role in the initiation of apoptosis. In muscle cells, ROS are produced in response to extracellular reactions or by cell mitochondria. It is, therefore, believed that mitochondria play a central role in apoptosis within skeletal muscle. Skeletal muscles have a well-developed system that protects them against oxidative damage. Myogenic stem cells are an integral part of multinucleated myofibers, and they are critically important for the maintenance of normal muscle mass, muscle growth, regeneration and hypertrophy. The latest research results indicate that myogenic cells are more sensitive to oxidative stress and pro-apoptotic factors than well-differentiated cells, such as myotubes. The complex structure and activity of skeletal muscle prompted research into the role of apoptosis and its intensity under various physiological and pathological conditions. This review summarizes the results of research investigating control mechanisms and the apoptosis process in skeletal muscle fibers, and indicates unresearched areas where further work is required.     

Nitrous oxide by itself is insufficient to relieve pain due to castration in piglets

Surgical castration is performed on most male piglets in the United States. However, castration is painful and analgesics have been considered to relieve pain. Inhalant gases with analgesic properties allow for a fast induction, have short-term and reversible effects, and are a needle-free option. Nitrous oxide (N(2)O; "laughing gas") has been widely used in human surgery and dental offices as an analgesic, sedative, and anxiolytic drug, yet N(2)O has not been thoroughly investigated for use in farm animals. We hypothesized that the analgesic effect of N(2)O could reduce the pain experienced by piglets during or immediately after castration. Twenty-four male piglets, from 12 litters, were castrated at 3 d of age. One piglet received N(2)O and a littermate received air as a control. After 150 s of exposure to the gas, castration was performed while the piglet remained exposed to the gas. Agitation scores and total vocalization length were recorded during castration. Behavioral observations were continued for 3 d postcastration by using a 5-min scan-sampling method for 4 h the first morning and for 2-h periods in the morning and afternoon of each day thereafter. Body weight gain was measured on the day before castration, at 3 d postcastration, and at weaning. Data were analyzed using a mixed model in SAS (Cary, NC). Nitrous oxide successfully induced anesthesia in all N(2)O piglets, as validated by a skin pinch test and the loss of the palpebral reflex. Total vocalization length was shorter in piglets receiving N(2)O during the induction phase (P = 0.003) but was not different during castration itself because piglets receiving N(2)O awoke and vocalized as much as control piglets (P = 0.87). Agitation scores during the whole procedure were reduced in piglets receiving N(2)O in both frequency (P = 0.005) and intensity (P = 0.026). For 2 h after castration, piglets receiving N(2)O displayed less huddling behavior than did control piglets (P = 0.01). Over the 3 d, piglets receiving N(2)O performed more tail wagging (P = 0.02) and tended to show fewer sleep spasms (P = 0.06) than did control piglets. Piglets given N(2)O tended to have a reduced growth rate compared with control piglets at 3 d postcastration and at weaning (P = 0.05 and P = 0.06, respectively). Nitrous oxide was effective in inducing anesthesia in neonatal piglets during handling. Nonetheless, its analgesic effects appeared insufficient in preventing castration-induced pain.     

大通牦牛骨骼肌低氧适应的组织学特点

选取2个海拔高度(3 200、3 700m)大通牦牛作为研究对象,以乐都地区(3 200m)牦牛为对照,利用光镜和计算机图像分析系统测定骨骼肌肌纤维直径、表面积密度;通过透射电镜比较骨骼肌线粒体的面数密度、体积质量、平均体积等结构参数。利用免疫组织化学技术测定骨骼肌组织血管内皮生长因子(Vascular endothelial growth factor,VEGF)和微血管密度(Microvessel density,MVD)。结果显示:(1)2个海拔高度的大通牦牛骨骼肌肌纤维直径均细于乐都牦牛骨骼肌肌纤维直径,差异显著(P0.05),肌纤维表面积密度,大通牦牛均大于乐都牦牛,差异显著(P0.05);(2)2个海拔高度的大通牦牛VEGF和MVD均高于乐都牦牛的VEGF和MVD,差异显著(P0.05);(3)海拔3 700m的大通牦牛骨骼肌线粒体平均体积、体积质量和面数密度均大于海拔3 200m的大通牦牛和乐都牦牛的平均体积、体积质量和面数密度,差异显著(P0.05)。结果表明:大通牦牛骨骼肌组织有良好的组织遗传学特性,表现出其育种父本的特征及对高原低氧环境的良好适应性,主要表现为肌纤维直径细、表面积密度大、骨骼肌组织VEGF表达量大和MVD大的特点。而骨骼肌线粒体的超微结构,更多的受环境因素的影响,主要表现为高海拔大通牦牛通过增加骨骼肌线粒体平均截面积、平均体积、体积密度和面数密度来提高其在低氧环境中对氧的利用。     

In vivo quantification of muscle damage in dogs after general anaesthesia with halothane andpropofol

Muscle damage in dogs anaesthetised with halothane and propofol was quantified by measurement of the area under the curve of plasma creatine kinase (CK) versus time. Plasma CK remained unchanged during anaesthesia for two and a half and five hours. Following halothane anaesthesia of dogs (resting on one side directly on the surgical table or on cushions, and with or without rotation of the body every 30 minutes), plasma CK was elevated in some animals to 10 000 U/litre by the 12th hour (baseline value ≤100 u/litre), whereas it remained almost unchanged in other animals. Plasma CK then returned to reference values on day 2 or 3. The mean equivalent of muscle damaged ranged from 0–6 to 0–9 g/kg bodyweight. No muscle damage could be demonstrated in animals anaesthetised with propofol. It is therefore concluded that plasma CK should not be used as a diagnostic aid following halothane anaesthesia because of false positives due to the halothane anaesthesia itself and that propofol Is best suited for the investigation of muscle damage due to surgical procedures.