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A portable lightweight stimulator for small animals is described. It delivers pulse trains of high intensity and is convenient for denervated muscle studies. It does not cause discomfort and does not restrict activity.
Neuronal nicotinic alpha7 subunits have been found in chick and rat skeletal muscle during development and denervation. In the present study, reverse transcriptase-polymerase chain reaction was used to detect alpha7 subunit mRNA in denervated mouse muscle. To determine whether the alpha7 subunit forms functional nicotinic acetylcholine receptors (nAChRs) in muscle, choline was used to induce a membrane depolarization because choline has been considered a specific agonist of alpha7-containing (alpha7*) nAChRs. We found, however, that choline (3-10 mM) also weakly activates muscle nAChRs. After inhibiting muscle nAChRs with a specific muscle nAChR inhibitor, alpha-conotoxin GI (alphaCTxGI), choline was used to activate the alpha7* nAChRs on muscle selectively. Four weeks after denervation, rapid application of choline (10 mM) elicited a substantial depolarization in the presence of alphaCTxGI (0.1 microM). This component of the depolarization was never present in denervated muscles obtained from mutant mice lacking the alpha7 subunit (i.e. alpha7-null mice). The depolarization component that is resistant to alphaCTxGI was antagonized by pancuronium (3-10 microM) and by a 4-oxystilbene derivative (F3, 0.1-0.5 microM) at concentrations considered highly specific for alpha7* nAChRs. Another selective alpha7 antagonist, methyllycaconitine (0.05-5 microM), did not strongly inhibit this choline-induced depolarization. Furthermore, the choline-sensitive nAChRs showed little desensitization over 10 s of application with choline (10-30 mM). These results indicate that functional alpha7* nAChRs are significantly present on denervated muscle, and that these receptors display unusual functional and pharmacological characteristics.
The total lipids triglycerides, free fatty acids, glycerol and acetoaectate levels in the gastrocnemius muscle of frog denervated for 1 month were compared. A significant atrophy without any change in total DNA content per whole muscle was noted on denervation. No significant change in the mitochondrial protein content in the atrophied muscle was noted. Relative to total muscle mass, the total fat, triglyceride, free fatty acids and glycerols decreased on denervation. This suggests the increased degradation of fat during denervation. Concomittantly lypolytic and esterase activities in the atrophied muscle increased. Palmitate and pyruvate oxidations in the mitochondria of atrophied muscle and also the enzymes of beta-oxidative scheme increased. 14C-acetate incorporation rates revealed that there is a decrease in fatty acid synthesis in the atrophied muscle. It was suggested that the increased fatty acid oxidation and decreased fatty acid synthesis in the atrophic process may not be the result of a simple functional demand; but may involve more factors in terms of neuro-muscular functions.
Concentration of Zn, Br, and Rb in intact and denervated rat muscles was estimated by X-ray fluorescent analysis. Differences in the content of these elements at different times after denervation were found. Concentration of microelements (microgram/g dry weight) in denervated muscles was shown to increase by the end of the third week, while that in the intact muscle decreased. The analysis of microelement content in the intact muscle revealed two steps of postdenervational changes: hypertrophic and atrophic ones. Differences between the above changes in microelement concentrations and their content in the intact muscle suggest a hypothesis that protein dissociation and utilization of microelements in muscle cells after denervation proceeds with different rates.
The sympathomimetic agent, clenbuterol, induces a muscle-specific hypertrophy in both normal and catabolic muscle. Drug-induced hypertrophy is not generally associated with an increase in DNA content, thus the role of satellite cells in the response of soleus muscles from weanling rats is questioned. Following simultaneous sciatic section and administration of clenbuterol, responses are similar in innervated and denervated muscles after 4 days. Increased protein accretion in treated muscles is associated with evidence of satellite cell activation, but with little evidence of division. It is speculated that satellite cell production of growth factors may play an important role in the hypertrophic action of clenbuterol, and the clinical implications of the findings are discussed.
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DNA synthesis in skeletal muscle increases dramatically during the first week after denervation. In the present study, we have characterized the dividing cells in order to assess the specificity and significance of this response to denervation. Autoradiography of [3H]thymidine-labeled denervated muscles revealed that many classes of cells were dividing, including fibroblasts (the most numerous of the labeled cells), macrophages, vascular cells, muscle satellite cells, spindle capsule cells, perineurial cells and Schwann cells. The number of labeled satellite cells accounted for no more than 10% of the dividing cells. Labeling indices of spindle capsule, perineurial and Schwann cells reached a maximum 3 days after denervation, while those of fibroblasts and macrophages peaked at 4 days. The former group of cells, which are in close contact with nerve trunks, accounted for 28% of cell division on day 3 (but only 5% on day 4) and were apparently responding to a local influence from degenerating axons. Connective tissue cells, making up the largest class of dividing cells (80% on day 4) were found throughout the muscle and appeared to proliferate in response to changes occurring along the entire length of the muscle fibers. Macrophages involved in the response were mostly resident histiocytes, since prior labeling of blood cells showed that leukocytes did not enter the muscle in substantial number after denervation. Both muscle satellite cells and connective tissue cells are essential for the functional regeneration of muscle. Thus, on the basis of overall cytologic characterization, cell division after denervation seems to represent a limited regenerative response.
The topological positions of nerve terminal sprouts in partially denervated rat sternocostal muscles were analysed. Sprouted nerve terminals were found only within 200 microns of a denervated muscle fibre, but were not necessarily within 200 microns of a denervated endplate. From this it was concluded that the sprout-inducing factor released from denervated muscle fibres does not necessarily arise from the denervated endplate, but may be released along the length of the muscle fibre. It is proposed that sprout factor is released during the incorporation of acetylcholine receptors into the muscle fibre membrane.
OBJECTIVE: To determine the sensitivity of the combined measurement of chronaxie and the accommodation index in the qualitative diagnosis of muscle denervation with needle electromyography and to compare quantitative diagnoses. DESIGN: Ninety-three neurogenic muscles diagnosed by needle electromyography were consecutively included for measurement of chronaxie and the accommodation index in this prospective study. The sensitivity of qualitative diagnosis was assessed for all muscles, separately for the acute and subacute-chronic denervation phase and for the complete and partial denervation. RESULTS: The combined measurement of chronaxie and the accommodation index showed a 90% sensitivity to needle electromyography for qualitative diagnosis of muscle denervation for all muscles. A 100% sensitivity was found for the acute denervation phase and for complete denervation. The subacute-chronic denervation phase revealed a 86% sensitivity, and partial denervation had a 88% sensitivity. The kappa coefficients did not show satisfactory agreement in quantitative diagnosis, and Bowker's test revealed a statistically significant underestimation of muscle denervation for measurement of chronaxie. CONCLUSION: The combined measurement of chronaxie and the accommodation index can be recommended for the screening of neurogenic lesions in the acute denervation phase.
The reported anabolic action of some beta 2 agonists may have clinical applications in certain muscle wasting states. Administration of clenbuterol (2 mg/kg diet for 14 days) to rats resulted in a limited degree of hypertrophy of normal muscles; the effect was more pronounced on fast-twitch muscles than on slow-twitch muscles. The anabolic effect was greatest in denervated muscles, where it was significantly more effective on the slow-twitch type. Clenbuterol significantly improved the contractile properties of denervated slow-twitch muscle, reverting them toward normal, but had little effect on contractile properties of denervated fast-twitch muscle. Such differential effects of clenbuterol must be taken into consideration in the evaluation of any future human intervention study.
In recent years a number of studies have employed long pulse biphasic stimulation as a treatment for denervated muscle to improve tissue quality and in some cases to improve contractile capability sufficient to restore function. However, in the U.K., this treatment is yet to be widely adopted clinically. A 5 subject, case based pilot study of long pulse biphasic direct stimulation of peripheral limb denervated muscle is being conducted and its effect on the tissue evaluated by measurement of muscle bulk, limb blood flow, and skin temperature. In cases of partial denervation. trapezoidal shaped pulses are used to minimize sensory and motor nerve fiber recruitment.
The author investigated the possibility and the mechanism of reinnervation of "endplate-free denervated muscle" by direct neurotization, using the anterior tibial muscles of rats. The proximal stump of the posterior tibial nerve, which was severed at the ankle level, was embedded into the transected distal 1/5 (5 mm) of the endplate-free segment of the muscle. M-waves by electromyogram appeared 6 weeks after neurotization. In the histological specimens with acetylcholine-esterase (Ach-E) stain, accumulations of Ach-E activity were detected from one week after neurotization. These increased gradually in both number and size. In the electron microscopy specimens, axons were seen in the synaptic fold-like indentations of the thickened sarcoplasm in 4 weeks. Eight weeks after neurotization, synaptic vesicles were observed in the axons. It was established by this study that direct neurotization resulted in the formation of new motor endplates and in the reinnervation of "endplate-free denervated muscle".
Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.
Transcriptional activity of acetylcholine receptor subunit genes was investigated in innervated and denervated chick skeletal muscle. The sciatic nerve of 3-d-old White Leghorn chicks was sectioned unilaterally; after various intervals, nuclei were isolated from operated and sham-operated animals, and run-on assays performed. Nuclei were incubated with 32P-UTP, and total RNA was extracted and hybridized onto filters containing an excess of subunit-specific DNA. Specific transcripts were detected by autoradiography and quantitated densitometrically. A sharp increase in transcriptional activity was observed to begin approximately 1/2 d after the operation and peak 1 d later when transcriptional rates reached approximately seven-, six-, and fivefold control levels for the alpha-, delta-, and gamma-subunit genes, respectively. The specificity of the effect was ascertained by normalization to total RNA synthesis and by the demonstration that several nonreceptor genes respond differently to denervation. These results suggest that a denervation signal reaches the genome to induce receptor expression. In addition, since the increase in mRNA levels significantly exceeds what can be accounted for by increased gene activity, posttranscriptional effects are suggested.
Although denervating injuries produce severe atrophic changes in mammalian skeletal muscle, a degree of functional restoration can be achieved through an intensive regime of electrical stimulation. An implantable stimulator was developed so that the long-term effects of different stimulation protocols could be compared in rabbits. The device, which is powered by two lithium thionyl chloride batteries, is small enough to be implanted in the peritoneal cavity. All stimulation parameters can be specified over a wide range, with a high degree of resolution; in addition, up to 16 periods of training (10-180 min) and rest (1-42 h) can be set in advance. The microcontroller-based device is programmed through a bidirectional radiofrequency link. Settings are entered via a user-friendly computer interface and annotated to create an individual study protocol for each animal. The stimulator has been reliable and stable in use. Proven technology and rigorous quality control has enabled 55 units to be implanted to date, for periods of up to 36 weeks, with only two device failures (at 15 and 29 weeks). Changes in the excitability of denervated skeletal muscles could be followed within individual animals. Chronaxie increased from 3.24 +/- 0.54 ms to 15.57 +/- 0.85 ms (n = 55, p < 0.0001) per phase in the 2 weeks following denervation.
Insulin signaling was examined in muscle made insulin resistant by short-term (24-h) denervation. Insulin-stimulated glucose transport in vitro was reduced by 28% (P < 0.05) in denervated muscle (DEN). In control muscle (SHAM), insulin increased levels of surface-detectable GLUT-4 (i.e., translocated GLUT-4) 1.8-fold (P < 0.05), whereas DEN surface GLUT-4 was not increased by insulin (P > 0.05). Insulin treatment in vivo induced a rapid appearance of phospho[Ser(473)]Akt-alpha in SHAM 3 min after insulin injection. In DEN, phospho[Ser(473)]Akt-alpha also appeared at 3 min, but Ser(473)-phosphorylated Akt-alpha was 36% lower than in SHAM (P < 0. 05). In addition, total Akt-alpha protein in DEN was 37% lower than in SHAM (P < 0.05). Akt-alpha kinase activity was lower in DEN at two insulin levels tested: 0.1 U insulin/rat (-22%, P < 0.05) and 1 U insulin/rat (-26%, P < 0.01). These data indicate that short-term (24-h) denervation, which lowers insulin-stimulated glucose transport, is associated with decreased Akt-alpha activation and impaired insulin-stimulated GLUT-4 appearance at the muscle surface.
OBJECTIVE: Magnetic resonance spectroscopy (MRS) has the potential to noninvasively delineate early biochemical changes in denervated muscle. In this study, we examine metabolic changes in denervated rat facial muscles using quantitative invitro 1H and 31P MRS. METHODS: Forty male Wistar rats were subjected to transection of the facial nerve trunk on the left and sham exposure on the right, and allowed to recover. The animals were then reoperated at 1, 2, 4, or 8 weeks after the initial procedure. EMG of the facial muscles and facial nerve conduction studies were performed at both time points, and facial muscles were harvested from normal and control sides at the second procedure. Perchloric acid extracts of facial muscles were then prepared for analysis using MRS. RESULTS: The results showed a progressive time-dependent decrease in Cr, PCr, Pi, ATP, and ADP all on the transected side. CONCLUSION: This study is an important step in the development of clinically relevant noninvasive methods of assessing and quantifying degeneration in nerve-muscle systems.
The evaluation of peripheral nerve disorders has traditionally relied on a clinical history, physical examination, and electrodiagnostic studies. Recent studies have used magnetic resonance imaging (MRI) to evaluate a variety of both nerve and muscle disorders. In this article, we describe the use of MRI, using short-tau inversion recovery (STIR) sequences, to evaluate muscle signal characteristics in a variety of peripheral nerve disorders. A total of 32 patients were studied, and 12 representative cases are discussed in detail. Increased STIR signal in muscle was seen in cases of severe axonotmetic injuries involving the transection of axons producing severe denervation changes on electromyography. The increased STIR signal in denervated muscles was seen as early as 4 days after the onset of clinical symptoms, which is significantly earlier than changes detected on electromyography. The MRI signal changes were reversible when the recovery of motor function occurred as a result of further muscle innervation. In cases of neurapraxic nerve injuries, characterized by conduction block without axonal loss, the STIR signal in muscle was normal. These findings show that MRI using STIR sequences provides a panoramic visual representation of denervated muscles useful in localizing and grading the severity of peripheral nerve injury secondary to either disease or trauma. MRI using STIR sequences may therefore play an important role in the prediction of clinical outcome and the formulation of appropriate therapy early after peripheral nerve injury.