Early changes in experimental denervated rat gastrocnemius muscle

Summary Computer-aided image analysis of EM images reveals important morphometric alterations after only 10 days of rat gastrocnemius denervation. The increasing importance of early quantitative changes in human diagnostics is stressed.Supported by FGWO grant no. 3.0004.82     

Stretching and electrical stimulation regulate the metalloproteinase-2 in rat denervated skeletal muscle

Abstract

Objective: To evaluate the effects of electrical stimulation (ES), stretching and their combined effects in matrix metalloproteinases (MMPs) gene expression and activity during early denervation of rat tibialis anterior (TA) muscle by axonotmesis.

Methods: Thirty adult male Wistar rats were divided into five groups: normal (N), denervated TA (D), denervated TA submitted to daily ES (D + ES), denervated TA submitted to daily stretching (D + St) and denervated TA submitted daily to both ES and stretching concurrently (D + ES + St). Both zimographic analysis and real time polymerase chain reaction of MMPs were used to muscular evaluation. In addition, muscle fiber cross-section area (CSA) was also evaluated.

Results: Stretching increased MMP-2 activity in denervated muscle when performed alone as well as in association with ES (p<0·01). Both stretching and ES, individually and in association, increased MMP-2 gene expression in denervated muscle compared to N and D (p<0·05). All denervated groups decreased muscle fiber CSA compared to N (p<0·05).

Discussion: While stretching is the main stimulus leading to the activation of MMP-2, both ES and stretching are able to increase MMP-2 gene expression in rat denervated muscle suggesting ECM remodeling.     

Effects of intermittent high frequency electrical stimulation on denervated EDL muscle of rabbit

This study was performed to determine whether electrical stimulation can retard denervation-induced changes. The denervated extensor digitorum longus of the rabbit, a fast-twitch muscle, was stimulated at a rate mimicking its motoneuron firing pattern. The 100-Hz stimulation given intermittently subjected the muscle to a low mean total daily frequency of 1.6 Hz. Four weeks of stimulation resulted in no effect upon the denervated stimulated muscle. This stimulation protocol, therefore, is unable to substitute for the lost neuronal influence of the nerve. The muscle contralateral to the stimulated side showed physiological changes making it unsuitable to serve as a control.     

The effect of isometric short-term electrical stimulation on denervated muscle

Electrical stimulation was applied daily for 20 minutes to denervated rabbit extensor digitorum longus muscle. One group was stimulated with short tetani, another with 1-Hz frequency, using isometric contractions for both. Tetanic stimulation induced severe fibrosis and is harmful to denervated muscle. One Hertz stimulation retarded denervation-induced fatigue and atrophy, as well as slowing of contraction time.     

Effects of long-impulse electrical stimulation on atrophy and fibre type composition of chronically denervated fast rabbit muscle

Summary The efficacy of electrical stimulation on a chronically denervated muscle depends on stimulus parameters, which have an important influence on the development of atrophy. Stimulus frequency and/or total activity are particularly responsible for the development of some histological, biochemical and contractile features. The present study in 18 rabbits deals with a recently developed electrical stimulus, which had proved effective in maintaining muscle force following denervation. This current has (1) unusual long bidirectional rectangular impulses (20 ms) and (2) a frequency of 25 Hz, which is between the frequencies of fast- and slow-firing motor units. Electrical stimulation began 28 (in one animal 53) days after total motor and sensory denervation of the right hindleg, and was continued until the end of the experiment, up to 205 days. To mimic a therapeutic regimen, which should be agreeable to patients, daily treatment times were kept to a minimum (2 × 6 min), and surface electrodes were used. Morphometric evaluation of the fast flexor digitorum sublimis muscle showed that such electrical stimulation was able to preserve fibre diameter at a level of 72–86% of the initial values for several months, while unstimulated fibres showed the usual atrophy with a decrease of diameters below 40% of normal. The stimulation induced a hybrid fibre type with properties of a slow muscle (rich in mitochondria in NADH-dependent tetrazolium reductase staining and electron microscopy) as well as of a fast-twitch muscle (fibre type IIb in myofibrillar ATPase stainings).     

The effect of low-frequency electrical stimulation on the denervated extensor digitorum longus muscle of the rabbit

Both extensores digitorum longi (EDL) muscles of rabbits were denervated by crushing the common peroneal nerves. The EDL muscle on one side was directly stimulated at 10-12 Hz via implanted electrodes. This treatment reduced the changes of twitch/tetanus ratios produced by denervation and prevented the slowing of contraction and relaxation that follows denervation. It is concluded that the stimulation reduced the duration of the active state of denervated muscles.
These effects of stimulation were reduced after 5 weeks, probably because by that time the slowing effect of low-frequency activity on the fast muscles became apparent.     

Influence of isaxonine on the target phenomenon,muscle fibre size and neuromuscular junction in the tenotomized and denervated gastrocnemius muscle of the rat

Summary Isaxonine is reported to accelerate the rate of peripheral nerve regeneration, stimulate axonal sprouting and promote motor and sensory function recovery. The target phenomenon in muscle fibers is the morphological expression of increased stretch activity in a diseased muscle. It can be produced in the gastrocnemius muscle of the rat by tenotomy and is inhibited by simultaneous denervation. The influence of isaxonine on this experimental model was studied.Twenty Wistar rats were submitted to bilateral section of the Achilles tendon and section of the left sciatic nerve. In ten rats, treated with isaxonine, the qualitative and quantitative changes in the gastrocnemius muscle were compared with the control group over a period of 3 weeks. No statistical differences were observed in the occurrence and inhibition of the target phenomenon, the muscle fibre size and the neuromuscular junction. Isaxonine has no direct effect on a diseased muscle with an intact nerve supply, nor in the early stages afte complete denervation.Supported by grant no. 3.000.4.82 from FGWO     

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。     

Effects of actinomycin D on contractile properties of denervated rat skeletal muscle

We studied the mechanical and electrical properties in the extensor digitorum longus muscle of Wistar rats in vitro 4 days after denervation. In a group of animals, actinomycin D was injected intravenously at the time of denervation. The prolongation of contraction and relaxation time, and the increase in twitch tension observed in denervated muscles were prevented by the drug. Other denervatory changes such as the elimination of cooling potentiation, posttetanic potentiation, staircase phenomenon, and shortening of caffeine contracture were also inhibited or partially reverted by actinomycin D. In addition, some electrical denervatory changes such as the decrease of resting membrane potential and slowing of maximum rate of fall of the action potential were partially abolished by actinomycin D. These results support the hypothesis that a new synthesis of proteins is related to denervation changes.     

Improved motor response due to chronic electrical stimulation of denervated tibialis anterior muscle in humans

Nine patients with complete denervation of the tibialis anterior were admitted to a stimulation program in order to restore dorsiflexion of the foot. Best results were obtained with pulses of 20-msec pulse width and 20-msec interval. After 3 weeks of training for 2 X 20 min/day, dorsiflexion due to stimulation was increased in all patients. In some of them, gait could be improved during the swing phase using electrical stimulation. The applied training program thus reversed the course of disuse atrophy and proved the feasibility of functional electrical stimulation for patients with denervated muscles.     

Responses to electrical stimulation of denervated human muscle fibres recorded with single fibre EMG.

Denervated muscle fibres were stimulated electrically with needle electrodes introduced close to a recording single fibre electrode. The denervated muscle fibre could be driven with rates up to 100 Hz. The jitter was large at threshold but low at suprathreshold stimulus strength. There was evidence of discrete low threshold sites along the denervated muscle fibre, seen as stepwise latency change on smoothly changing stimulus strength, hepatic activation from other fibres and also as extra-discharges originating from such sites.     

Elevation of serum prostagladin E levels following electrical stimulation of the midbrain

Serum prostaglandin E(PGE) levels were measured in rats immediately following electrical stimulation of the mesencephalic periaqueductal gray (PAG) region and the nucleus raphe magnus (NRM) and compared to controls. An additional group received aspirin prior to PAG stimulation. A significant increase in serum PGE levels was found after stimulation of the PAG, but not the NRM. Aspirin inhibited the stimulation-induced increases in PGE.     

大鼠肌源性干细胞移植对失神经腓肠肌萎缩进程的影响

背景：目前，国内外有关肌源性干细胞移植的研究主要集中在治疗肌营养不良性萎缩症方面，尚未见涉及将肌源性干细胞应用于失神经肌萎缩综合治疗的报道。 目的：探讨大鼠肌源性干细胞移植对失神经腓肠肌萎缩进程的影响。 设计、时间及地点：随机对照动物观察，于2007-02/2008-07在沈阳医学院完成。 材料：成年健康雄性Wister大鼠19只，由沈阳医学院实验动物中心提供。 方法：取3只大鼠，无菌条件下切取肱三头肌，体外分离培养肌源性干细胞，贴壁法纯化扩增。余16只大鼠均切断右后肢胫神经，建立失神经腓肠肌模型，分为2组：细胞移植组于已切断的胫神经支配的腓肠肌的内外侧注射经DAPI标记的肌源性干细胞悬液0.2 mL，模型对照组于相同位置注射等量生理盐水，培养4周。 主要观察指标：采用计算机生物信号采集处理系统测定反映肌张力的腓肠肌阈强度恢复率和最适强度恢复率变化，通过电子天平计算肌湿质量残存率，应用图像分析系统测定肌纤维横截面积残存率。 结果：与模型对照组比较，细胞移植组失神经腓肠肌的阈强度恢复率、最适强度恢复率均明显降低(P < 0.01，P < 0.05)，肌湿质量残存率、肌纤维横截面积残存率均明显升高(P < 0.01)。 结论：肌源性干细胞移植对大鼠失神经肌萎缩进程有较明显的延缓作用。     

Electrical stimulation based on chronaxie reduces atrogin-1 and myoD gene expressions in denervated rat muscle

Denervation induces muscle fiber atrophy and changes in the gene expression rates of skeletal muscle. Electrical stimulation (ES) is a procedure generally used to treat denervated muscles in humans. This study evaluated the effect of ES based on chronaxie and rheobase on the expression of the myoD and atrogin-1 genes in denervated tibialis anterior (TA) muscle of Wistar rats. Five groups were examined: (1) denervated (D); (2) D+ES; (3) sham denervation; (4) normal (N); and (5) N+ES. Twenty muscle contractions were stimulated every 48 h using surface electrodes. After 28 days, ES significantly decreased the expression of myoD and atrogin-1 in D+ES compared to the D group. However, ES did not prevent muscle-fiber atrophy after denervation. Thus, ES based on chronaxie values and applied to denervated muscles using surface electrodes, as normally used in human rehabilitation, was able to reduce the myoD and atrogin-1 gene expressions, which are related to muscular growth and atrophy, respectively. The results of this study provide new information for the treatment of denervated skeletal muscle using surface ES.     

Mechanical and electrical properties of denervated rat skeletal muscles

Mechanical activity (twitch and tetanus) and electrical activity (single and repetitive action potentials) were recorded in vitro (34 degrees C) in control and denervated (3 to 14 days) soleus and extensor digitorum longus muscles of the rat. After denervation tetanic tension (100 to 200 Hz, 500 ms duration) was decreased in both types of muscles. Denervation reduced significantly the rates of rise and fall and the amplitude of the action potential in both types of muscle fibers. In denervated fibers with very low resting membrane potential no action potentials could be recorded: in these fibers only a slow response without overshoot was detected. Hyperpolarization of denervated fibers to -90 mV prior to application of the depolarizing pulse increased their excitability. Action potential amplitudes were well maintained during tetanic stimulation (200 Hz, 40 to 90 ms) in innervated fibers. Depolarization of the innervated fibers with cathodic current before the tetanic pulse hindered the generation of repetitive action potentials at 200 Hz. A proportion of denervated fibers stimulated at 100 to 200 Hz generated only one action potential or gave rise to an incomplete train. Hyperpolarization of the denervated fibers resulted in an improvement in the ability to generate a train of action potentials at 100 to 200 Hz. A group of denervated fibers exhibited well maintained action potentials during tetanus. We suggest that failure in the repetitive electrical activity of denervated fibers could be the reason for the reduced tension of tetanus. Depolarization of the fibers and/or the increment in the electrical time constant of the sarcolemma are suggested for the decrease in the electrical excitability of denervated fibers.     

Conditioning lesion effects on rat sciatic nerve regeneration are influenced by electrical stimulation delivered to denervated muscles

The rate of regeneration and the initial delay of the fastest growing fibers of the rat sciatic nerve were electrophysiologically evaluated after a freeze at mid-thigh. A prior section or a prior freeze at the ankle level increased the rate of regeneration and decreased the initial delay with different magnitudes. These phenomena are named ‘conditioning lesion effects’. A daily electrical stimulation transcutaneously delivered to the foot sole muscles from the day following their denervation by the prior lesion did not modify the increased rate of regeneration but prevented the decrease of the initial delay whatever the type of the prior lesion. Therefore, the initiation of earlier sprouting of the parent axons seems to be specifically controlled by a signal associated with muscle denervation properties.     

Motor endplate position of rat gastrocnemius muscle

In this study, the relative endplate position of fibers of rat gastrocnemius caput mediale (GM) muscle was determined by counting numbers of sarcomeres. Isolated fibers were teased from the proximal, intermediate, and distal regions of the muscle. Endplates of distal fibers were located on the proximal third of their lengths. Endplates of intermediate fibers were located at half fiber length, and for proximal fibers, a variable endplate position was obtained: in half of the muscles studied, endplates occurred around the proximal one-third and in the other half near the midpoint of the fiber. Endplate position relative to fiber length was thus found to be dependent on the region of the muscle. Changes in the orientation of endplate zone relative to the muscle belly is likely to take place with changes in muscle length, as shown by a planimetric muscle model. It is argued that architecture of pennate muscles may highly affect characteristics of motor unit potentials.