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Electron microscopic-cytochemical and biochemical studies of acetylcholinesterase activity in denervated muscle of rabbits.

Acetylcholinesterase (AChE) activity has been studied in normal, control and denervated muscle of rabbits by electron microscopic-cytochemistry and radiometric assay. A small amount of butyrylcholinesterase (BuChE) activity is also found in biochemical assay of unfixed muscle, but it is not demonstrable cytochemically in fixed specimens by the method used in this study. Both a soluble and particulate AChE activity are present in all specimens examined. The particulate activity is probably due to enzyme localized in the sarcotubular system and at the motor end-plate. Soluble AChE activity may represent those sites exhibiting random cytochemical end product, such as some areas of normal and denervated muscle and muscle nuclei, Schwann cells, and AChE-containing mononuclear cells in the connective tissue. There is a greater proportion of particulate than soluble AChE activity in normal and control muscle, a finding which is compatible with the well localized cytochemical sites. Four to six weeks post-denervation, there is a marked increase in extrajunctional AChE activity to peak values 15 to 30 fold above control values. The increase is accompanied by a reversal in the proportion of particulate to soluble enzyme, so that there is almost twice as much soluble as particulate AChE. There are also numerous "spots" of random cytochemical end product throughout extrajunctional muscle. The increase in levels of AChE activity, the change to predominantly soluble form, and the large numbers of new cytochemically active sites indicate that synthesis of new enzyme has taken place. Changes in AChE activity in denervated rabbit have been compared to those occurring in dystrophic mouse muscle. It has been suggested that there might be a relationship between the formation of new extrajunctional sarcoplasmic sites of AChE activity and the spread of alpha-bungarotoxin binding sites and chemosensitivity in developing and denervated muscle.

Acetylcholinesterase↗

Enhanced stimulation of diacylglycerol and lipid synthesis by insulin in denervated muscle. Altered protein kinase C activity and possible link to insulin resistance.

Denervated muscle is generally regarded as insulin resistant because the ability of insulin to stimulate glucose transport and glycogen synthesis is impaired. Previous studies indicate that insulin resistance in these muscles is likely due to a defect at a postreceptor site in the signaling pathway. Because glucose transport into cells has been reported to be linked to changes in diacylglycerol (DAG) and protein kinase C (PKC), we investigated the effect of denervation on the content and synthesis of DAG and the activity and distribution of PKC in the soleus muscle. The DAG content in muscles denervated for 24 h was 40% greater than in control muscles. This was associated with a two- to threefold increase in the percentage of total PKC activity that was membrane associated, with no significant change in total PKC activity, suggesting an increase in PKC activity in vivo. Studies of glucose disposition confirmed that the stimulation of glycogen synthesis by insulin and, to a lesser extent, 2-deoxyglucose uptake were impaired by denervation. However, the stimulation by insulin of glucose incorporation into DAG and other lipids was two- to threefold greater in denervated than in control muscles, and conversion of glucose to lactate and pyruvate and glucose oxidation to CO2 were unchanged. The results reveal a dichotomy in the effects of denervation on various actions of insulin, with both insulin resistance and hyperresponsiveness occurring in different pathways of glucose metabolism. They also reveal a potential mechanism for the elevation of muscle DAG after denervation. The results do not support a direct link between DAG-PKC and glucose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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.

Animals↗

Adrenalectomy eliminates both fiber-type differences and starvation effects on denervated muscle.

This report describes changes in muscle mass of innervated and denervated pairs of muscles taken from intact and adrenalectomized 250-g male Sprague-Dawley rats provided with different diets. Diets ranged from a nutritionally complete liquid diet to starvation (water only). In the intact animals, muscles with a more tonic character (soleus) are less sensitive to starvation than are muscles with a more phasic character (extensor digitorum longus), whereas the opposite is true of denervation. In the intact animals, starvation greatly increased the amount of atrophy following denervation. In the adrenalectomized animals, starvation had no effect on the amounts of atrophy following denervation. Furthermore, adrenalectomy virtually eliminated the fiber-type differences in the amount of atrophy following denervation. In addition, a comparison between denervated muscles from intact animals and adrenalectomized animals subjected to starvation demonstrates that all denervated muscles from the adrenalectomized animals atrophy less. Finally, it was observed that although an adrenalectomized animal can tolerate 6 days of starvation, an adrenalectomized-castrated animal cannot tolerate even short periods of starvation. The difference appears to be due to low amounts of corticosterone of testicular origin.

Adrenalectomy↗

[A successive study of histopathological changes in unilateral facial muscle denervation].

OBJECTIVE: To observe the histopathological changes in unilateral facial muscle denervation. METHODS: Gomori trichrome stain, acridine orange (AO) fluorescence, enzyme-histochemistry and immunohistochemistry techniques were used for studying mitochondria, nuclei acid concentration, enzymatic activity and contraction protein expression of facial muscle in different denervation period. RESULTS: Mitochondrian function, enzymatic activity and contraction protein expression of facial muscle denervation increased in the first two weeks, then declined till 6 months. Myofiber's histotype began to transform in 1 month after denervation. Type-grouping was obvious in 6 month. Regeneration myofibers were also seen at this time. CONCLUSIONS: Unilateral facial muscles become atrophy after denervation. They have proliferating tendency from 2 month to 6 month. Therefore, within 1 month after denervation measures to benefit atrophy will be more effective.

Animals↗

Maintenance, training and functional use of denervated muscles.

In the case of cerebral paralyses electrical stimulation can not only maintain the muscles, but may also enable their functional use. In flaccid paralyses, however, the conventional therapy using exponential currents produces rather unsatisfactory results. Only when applying bi-directional currents, were we successful in producing tetanic contractions. At present, some 20 children suffering from different diseases, such as spina bifida, Erb's palsy or a tumour of the cord, perform a daily domiciliary treatment with especially constructed home stimulators. Measurements prove distinct improvements of blood circulation, phosphoric metabolism and of the condition of the affected extremities. First investigations with computer-controlled, multi-channel devices show that by means of these devices the efficiency of training can be improved, the daily time for treatment can be shortened, and the disabled patient can perform the training almost autonomously. The existing experience on simple locomotion chains, and the achieved strengthening of the muscles will gradually enable a functional electrical stimulation of flaccid, denerved muscles and thus extend the radius of action for the disabled patient, for example by gripping, standing upright or walking.

Biomedical Engineering↗

Reinnervation of denervated muscle by transplantation of fetal spinal cord to transected sciatic nerve in the rat.

When motor neurons in the spinal cord are destroyed, regeneration of motor axons and muscle reinnervation cannot be expected. We attempted reinnervation of the denervated muscle, i.e. motor unit reconstruction, using transplantation of the fetal spinal cord to the peripheral nerve. The sciatic nerve of an adult rat was resected for 20 mm, and a cavity was prepared using an autologous femoral vein at the distal stump of the nerve. The fetal spinal cord was then transplanted into the venous cavity. After 3-6 months, no voluntary muscle contraction was observed due to the absence of communication with the central nervous system. However, reinnervation of the muscles via the sciatic nerve by the transplanted spinal neurons was demonstrated electrophysiologically and histochemically. This suggested that a motor unit can be reconstructed by fetal spinal cord transplantation even if the original motor neurons in the spinal cord are not available.

Animals↗

Mechanical properties of denervated amphibian muscle.

Denervated amphibian muscle does not show the prolongation of action potential found in mammalian denervated muscle. It was, therefore, predicted that denervated amphibian muscle would not show prolongation of the mechanical twitch. The sartorius muscles in one leg of toads--Xenopus borealis--were denervated for 140-268 days. Isometric twitch time to peak, time to half relaxation and twitch/tetanus ratio were not changed following denervation, confirming our prediction. Twitch tension decreased to 68% and tetanic tension decreased to 75% of control values. The maximum velocity of unloaded shortening (muscle length/s) was also unchanged.

Action Potentials↗

Electrical stimulation of denervated muscles: first results of a clinical study.

To evaluate the effects of electrical stimulation on denervated muscles in spinal cord injured humans, the EU Project RISE was started in 2001. The aims of this project are: to design and build sufficient stimulators; to develop stimulation protocols by means of mathematical models, animal experiments, and practice in humans with denervated lower limbs; to develop examination methods and devices for evaluation of electrical stimulation training effects; and to acquire basic scientific knowledge on denervated and stimulated denervated muscle. In the clinical study 27 spinal cord injured individuals were included, furthermore 13 pilot patients participated. After a series of initial examinations they underwent an electrical stimulation program for their denervated lower limb muscles. Some of the patients have already follow up examinations. A marked increase of muscle mass and quality was observed, the trophic situation of the denervated lower limbs had improved obviously.

Adult↗

Experimental study of neurotization of denervated muscles with nerve-to-vein transfer.

Neurotization of denervated muscles was attempted by means of coaptation of peripheral nerves to the veins draining these muscles. In Sprague-Dawley rats, the lateral gastrocnemius branch from the tibial nerve was severed and its proximal end was sutured to the distal stump of the lateral gastrocnemius vein. In all animals nerve-muscle communication was confirmed electrophysiologically 2 and 6 months after the operation. However, histological examination revealed that the regenerated nerve fibers were not within the vessel lumen, but ran through the scar tissue in close proximity to the outside of the vessel, to enter the muscle. Control animals, in which only nerve resection was done, did not display nerve reinnervation. These results suggest that, although nerve fibers did not regenerate into the vein, the vein works well as a guide for regenerating nerve fibers to denervated muscle.

Animals↗

Visualization of denervated muscle by gadolinium-enhanced MRI.

Thirty patients presenting with foot drop due to lesions of the peroneal nerve or L5 spinal root were investigated with gadolinium (Gd)-enhanced MRI of the lower leg. Significant enhancement was only seen in the denervated muscles in a pattern appropriate for the distribution of the nerve or root. In a rat model, identical changes in the denervated muscle were reproduced and seen as early as 24 hours after sciatic nerve transection. Thus, Gd-enhanced MRI is a new and sensitive technique to visualize denervated muscle.

Animals↗

[Ultrasound follow-up after experimental muscle denervation].

AIM: To describe sonographical results following acute, experimental muscle denervation. METHOD: Denervation of the supraspinatus and infraspinatus muscles was performed in 28 New Zealand white rabbits by segmental resection of the suprascapular nerve. The changes in the sonographic image of the muscles were follow up and documented at short intervals over 2 months. RESULTS: The sonographically detectable changes following denervation follow a definite pattern. In addition to the reduction in muscle diameter, sonographical signs of denervation include an increase of echodensity and an inhomogeneity of echotexture that appeared on day 14 after injury and became more prominent at larger intervals. CONCLUSION: Sonography may play a supportive role in the diagnosis and follow-up of neurogenic muscle atrophy.

Animals↗

Increase of Cardiotrophin-1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats.

The original report by Pennica et al. on Cardiotrophin-1 (CT-1) states that it markedly stimulates hypertrophy in cardiac myocytes both in vitro and in vivo and is predominantly expressed in the early mouse embryonic heart tube. CT-1 is a member of the interleukin-6 superfamily and past studies have shown that it exerts trophic effects on neurons, glial cells and their precursors, and is expressed during myogenesis. Thus CT-1 is associated with physical and pathological changes in skeletal muscle. In this study, we examined whether CT-1 is expressed in mechanically overloaded, regenerating, and denervated muscles of rats using immunohistochemistry. In the overloaded plantaris muscles at 1 and 3 days postsurgery, CT-1 immunoreactivity was detected in the mononuclear cells that had infiltrated the extracellular space. CT-1 immunoreactivity was also observed in the mononuclear cells invading the extracellular space at 2, 4, and 6 days after a bupivacaine injection and in degenerative and necrotic muscle fibers at 2 days postinjection. In the denervated muscles, the CT-1 immunoreactivity did not change in intensity during the entire period of the denervation (2, 7, and 14 days postsurgery). The cells invading extracellular space and in necrotic muscle fibers possessing CT-1 immunoreactivity might be muscle precursor cells (satellite cells) or migrating macrophages undergoing phagocytosis. Using double-immunostainings for anti-CT-1/antic-met, anti-CT-1/ anti-M-cadherin, and anti-CT-1/anti-ED1, we found that satellite cells and macrophages exhibited CT-1 immunoreactivity in the damaged muscles after bupivacaine injection. We therefore believe that CT-1 plays a key role in regeneration and hypertrophy in the skeletal muscle of rats.

Anesthetics, Local↗

[Effect of different allogenic cells injected into denervated muscles on nerve regeneration in rats].

OBJECTIVE: To study the effect of allogenic different cells injected into denervated muscles on nerve regeneration. METHODS: Thirty-six adult female SD rats, weighted 120-150 g, were divided into four groups randomly (n = 9, each group). Left sciatic nerves were cut down on germfree conditions and given primary suture of epineurium. Different cells were injected into the muscles of calf at once after operation every seven days and in all four times (group A: 1 ml Schwann cells at concentration of 1 x 10(6)/ml; group B: 1 ml mixed cells of Schwann cells and myoblast cells at concentration of 1 x 10(6)/ml; group C: 1 ml extract from the culture medium of kidney endothelial cells; and group D: 1 ml culture medium without FCS as control). After 3 months, the specimen was observed on macrobody and histology, and the densities of neurilemma cell and myoceptor were counted. RESULTS: The means of proximate neurilemma cells were 0.1877 +/- 0.0542 in group A, 0.1551 +/- 0.0321 in group B, 0.0724 +/- 0.0237 in group C, and 0.1877 +/- 0.0542 in group D. The densities of myoceptor were 6.000 +/- 0.866 in group A, 9.000 +/- 2.291 in group B, 12.780 +/- 1.394 in group C, 3.110 +/- 0.782 in group D. CONCLUSION: Schwann cells, mixed cells of Schwann cells with myoblast cells, and the extract from kidney endothelial cells can all accelerate the nerve regeneration. And the effect of extract from the kidney endothelial cell is superior to that of Schwann cell and mixed cell.

Animals↗

Satellite cells in denervated muscles.

It is known that, in a denervated striated muscle, the satellite cells multiply by mitotic division. A liaison between these satellite cells and the Schwann cell in front of the post-synaptic membrane in denervated frog muscle has been observed. It is probable that such cell connections help in the subsistence of the Schwann cell in a denervated muscle.

Animals↗

Electrical stimulation of denervated muscles of rats maintains mass and force, but not recovery following grafting.

PURPOSE: Denervated skeletal muscles lack contractile activity and subsequently lose mass and force generation. Prolonged periods of denervation prior to nerve-implant grafting limit the recovery of mass and force. We hypothesized that electrical stimulation during a period of denervation that maintains mass and force above the levels of denervated muscles enhances the recovery of mass and force following nerve-implant grafting. METHODS: The extensor digitorum longus (EDL) muscles of anesthetized rats were denervated, and a stimulator was implanted. Following 4 or 7 months of denervation, with or without electrical stimulation, the EDL muscles were removed, evaluated in vitro for mass and contractile properties, and then nerve-implant grafted into syngeneic rats. Unoperated, contralateral muscles were also evaluated and grafted. RESULTS: The hypothesis was not supported by the experimental data. Compared with values for 4- or 7-month denervated muscles, the stimulated-denervated muscles maintained higher mass and force, less prolonged time-to-peak tensions and half-relaxation times, and higher excitability. Nevertheless, the recovery of mass and force following grafting was not improved. CONCLUSION: The factors within long-term denervated muscles that hinder recovery following grafting appear to be related primarily to factors associated with the duration of denervation and not to the level of atrophy and weakness prior to grafting.

Animals↗

[Power measurement of denervated muscle isografts with neurorrhaphy and nerve-implantation in rats].

The hypothesis was tested in this experiment that after 7 months of predencervation, the reinnervation of muscle grafts with neurorrhaphy results in greater recovery of force and power than with nerve-implantation. In a highly inbred strain of rats, soleus muscles were isografted. The donor muscles were either immediately denervated at the time of isografting or denervated 2, 4, 7 months prior to isografting. Soleus muscles from each donor group were transplanted into the right legs of hosts with either epineurial anastomosis (NR group) or nerve implantation (NI group). The contralateral soleus muscles of hosts served as controls. Sixty days after transfer, both right and left soleus grafts/muscles were evaluated for force and power measured in situ. The absolute force values were significant higher in NR group (61% of normal) than in NI group (40% of normal) in 2-month group but the result invenrsted in 7-month group, less than 20% and more than 20% of normal in NR and NI groups respectively. The reduced ability of grafts to generate force and power resulted from the different ways of reinnervation in denervated muscles and the period of predenervation. Maybe the nerve-implantation is better than the neurorrhaphy for reinnervating a long-term denervated muscle.

Anastomosis, Surgical↗