Effects of acrylamide and some other sulfhydryl reagents on spontaneous and pathologically induced terminal sprouting from motor end-plates

The innervation of the normal rat sternocostalis muscle exhibits a constant low level of short spontaneous terminal sprouts visible in zinc iodide-osmium (ZIO) and in methylene blue-stained preparations. Acrylamide inhibits these spontaneous sprouts in a dose-dependent manner. This inhibition is mimicked by N-ethylmaleimide (a sulfhydryl group blocking agent) and can be nullified when acrylamide is given after the sulfhydryl group protecting agent, dithiothreitol (DTT). This could not be reversed by giving DTT 3 hours after acrylamide. Furthermore, when given alone DTT increases the level of spontaneous terminal sprouting seen in ZIO and in methylene blue-stained preparations. These findings suggest that the binding of acrylamide to sulfhydryl groups is involved in the inhibitory process. Acrylamide also reduces the number and length of the reactionary terminal sprouts, seen in ZIO and in methylene blue-stained preparations, that follow partial denervation or local injection of botulinum toxin. These inhibitory effects are long-lasting; recovery still has not fully occurred 4 weeks after a single dose of acrylamide (50 mg/kg). The roles of glutathione and other sulfhydryl components of axons are discussed in relation to the mechanism of acrylamide neurotoxicity.     

Regulation of Motoneuronal Calcitonin Gene–related Peptide (CGRP) During Axonal Growth and Neuromuscular Synaptic Plasticity Induced by Botulinum Toxin in Rats

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene–related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.     

Ciliary neurotrophic factor is required for motoneuron sprouting

We used mutant mice that lack the gene for ciliary neurotrophic factor (CNTF) to test the hypothesis that it is an endogenous sprouting factor. Fibers in the lateral gastrocnemius muscle were either partially denervated by transection of one of the branches of its nerve or paralyzed by intramuscular injection of botulinum toxin. This results in a significant sprouting response at the terminals of intact motoneurons in normal animals. We did not detect sprouting produced by either stimulus in mice lacking CNTF. When exogenous CNTF was administered to CNTF knockout mice following partial muscle denervation, they mounted a typical sprouting response. Thus CNTF is a critical factor in the process of sprout formation after both partial denervation injury and neuromuscular paralysis. It may function as part of a cellular compensatory mechanism after neuronal injury.     

Comparison of neuromuscular blockade and recovery with botulinum toxins A and F

Intramuscular injection of botulinum toxin A is used to treat focal dystonias. Because immunoresistance has been documented in some patients, other molecular forms of the toxin have been evaluated clinically. The present investigation compared the time course and extent of neuromuscular blockade and recovery of botulinum toxin types A and F using an electromyographic monitoring system implanted in the rat hindlimb. For a given dose, the degree and duration of blockade was more complete with type A toxin. Delayed onset of recovery in animals that received high doses of type A toxin allowed time for denervative changes to prevent a full return to baseline, as confirmed histologically. Conversely, animals receiving type F toxin fully recovered within 30 days at all dose levels. The rapid recovery with type F toxin suggested that neuromuscular transmission was restored via the original terminals rather than through functional collateral sprouting. The reversible nature of blockade with this molecular species puts in question its future clinical utility.     

Key changes in denervated muscles and their impact on regeneration and reinnervation

The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle＇s residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.     

Expanding use of botulinum toxin

Botulinum toxin type A (BTX-A) is best known to neurologists as a treatment for neuromuscular conditions such as dystonias and spasticity and has recently been publicized for the management of facial wrinkles. The property that makes botulinum toxin type A useful for these various conditions is the inhibition of acetylcholine release at the neuromuscular junction. Although botulinum toxin types A and B (BTX-A and BTX-B) continue to find new uses in neuromuscular conditions involving the somatic nervous system, it has also been recognized that the effects of these medications are not confined to cholinergic neurons at the neuromuscular junction. Acceptors for BTX-A and BTX-B are also found on autonomic nerve terminals, where they inhibit acetylcholine release at glands and smooth muscle. This observation led to trials of botulinum neurotoxins in various conditions involving autonomic innervation. The article reviews the emerging use of botulinum neurotoxins in these and selected other conditions, including sialorrhea, primary focal hyperhidrosis, pathological pain and primary headache disorders that may be of interest to neurologists and related specialists.     

Early degeneration and sprouting at the rat neuromuscular junction following acrylamide administration

Prolonged acrylamide administration produces motor nerve-terminal branch degeneration and impairs axonal outgrowth following nerve crush. It is unclear how early terminal branch degeneration is initiated and whether there is a compensatory regenerative response at the neuromuscular junction (NMJ). A modified Pestronk and Drachman silver-acetylcholinesterase strain was used to carry out a detailed morphometric analysis of the NMJ in soleus and lumbrical muscles. Rats were given 3, 5, or 10 doses of acrylamide, 35 mg/kg/day, by intraperitoneal injection, 5 days/week, and killed 4, 7, or 14 days after the first dose, respectively. Degenerating terminal branches were evident in soleus NMJ after only three doses of acrylamide. Diminished synaptic vesicle content, neurofilament accumulations and tubulo-vesicular profiles were evident after three doses. At later time points, degenerating terminals contained few synaptic vesicles and were engorged with neurofilaments. Endplate lengthening, indicative of denervation supersensitivity, accompanied degeneration. Terminal sprouting proliferated after 3 and 5 doses but was less prominent after 10 doses. Although similar changes occurred in the lumbrical muscle, they were not initiated until after 5 doses. These experiments reveal that pathological changes in terminal branches commence earlier and after a lower cumulative dose of acrylamide than previously reported and suggest that acrylamide exerts a primary effect at motor nerve-terminal branches. Early, vigorous terminal sprouting indicates that acrylamide does not prevent the initiation of regeneration, but with prolonged treatment does cause degeneration of maturing sprouts.     

Nerve stimulation boosts botulinum toxin action in spasticity.

Spasticity leads to functional and structural changes in nerves and muscles, which alter skeletal muscle function. To evaluate whether short-term electrical nerve stimulation (NS) improves the effect of botulinum toxin in spastic skeletal muscle, we studied changes in the amplitude of the compound muscle action potential (CMAP) recorded from the extensor digitorum brevis (EDB) muscle in response to peroneal nerve stimulation at the ankle after injection of botulinum toxin type A (BTXA) alone or combined with short-term NS. In paraparetic patients, both EDB muscles were injected with BTXA; and NS was applied to one EDB muscle alone. All patients received a 30-minute session of electrical NS once a day for 5 consecutive days after BTXA injection. We used two different stimulation frequencies (low-frequency, 4 Hz; and high-frequency, 25 Hz). EDB-CMAP amplitudes were evaluated before BTXA injection (day 0) and changes in CMAP amplitude, expressed as a percentage (CMAP%), were measured at various time points over a 30-day period after BTXA injection. We compared changes in the CMAP% amplitude on the stimulated and contralateral nonstimulated sides. We also studied the electromyographic activity recorded from EDB muscles over a 30-day period. CMAP% amplitudes measured at all time points after BTXA injections were significantly reduced in both EDB muscles. On days 4, 10, and 15, the CMAP% amplitude reduction was significantly greater for the low-frequency stimulated EDB than for the contralateral nonstimulated EDB. No significant differences in CMAP% were observed for the high-frequency stimulated and nonstimulated EDB. After BTXA injection, spontaneous activity appeared in both EDB muscles; but it appeared earlier and involved larger areas in the stimulated than in the nonstimulated EDB. In conclusion, short-term NS accelerates the effectiveness of intramuscular BTXA injections on the neuromuscular blockade in patients with spastic paraparesis and could induce a rapid and persistent improvement in spasticity. Its action probably arises mainly from low-frequency NS.     

Type grouping in skeletal muscles after experimental reinnervation: another explanation

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.     

Early terminal and nodal sprouting of motor axons after botulinum toxin

Axonal sprouting in distal motor axons was studied in an attempt to answer two questions: (a) is the cell body required for early axonal sprouting?, and (b) do nodal, as well as terminal, axonal sprouts arise after muscle inactivity not caused by nerve injury? Botulinum toxin (BT) was used to induce axonal sprouting without nerve trauma. Mice were injected in the right calf with a sublethal dose of BT and the soleus muscle examined ultrastructurally at times varying from 3 h to 5 days post-injection. Terminal axonal sprouts were seen 2 days after injection, and based on the time taken for BT to act and the growth rate of sprouts, axons were calculated to sprout within 24 h of muscle inactivity. This short time suggests that early axonal regrowth is initiated and controlled at the distal axon. Sprouts were seen arising from the intramuscular nodes of Ranvier from 2 days after BT injection. Unlike the terminal sprouts which elongated over time, the nodal sprouts remained short and confined by the basal lamina overlying the node, probably because without structural denervation there were no empty perineural sheaths to act as pathways to the motor endplates. The finding of terminal and nodal sprouts after botulinum toxin supports the hypothesis that muscle inactivity gives rise to a single growth factor for both terminal and nodal sprouting.     

Costimulation blockade in transplantation of nerve allografts: long-term effects

Costimulation blockade can prevent rejection of nerve allografts in short-term studies. We tested if costimulation blockade also prevented rejection of nerve allografts in long-term experiments, thereby improving functional recovery. A 7-mm sciatic nerve defect in C57/BL6 mice was bridged either by nerve allografts from Balb/C mice or by isogenic nerve grafts (isografts) from C57/BL6 mice. Costimulation blockade in the form of a triple treatment with anti-LFA-1, anti-CD40L, and CTLA4Ig was given at post-operative days 0, 2, 4, and 6 (intraperitoneal). Control mice (placebo; allografts) with nerve grafts were treated with isotype antibodies during the same time period. After 49 days, tetanic muscle force, wet weight of gastrocnemius muscle, histology, and morphometry in the tibial nerve were evaluated. Costimulation blockade diminished rejection of the nerve allografts. Axons bridged the graft. Treatment increased wet weight of the gastrocnemius muscle and resulted in a higher mean myelin area/nerve fiber in the tibial nerve distal to the nerve grafts. Tetanic muscle force and number of axons in tibial nerve showed no differences between groups. We conclude that rejection is suppressed by costimulation blockade. Treatment improves recovery of target muscle and myelination after nerve allografting.     

Nerve growth from nodes of Ranvier in inactive muscle

Paralysis of mouse gluteus maximus muscle with botulinum toxin not only evoked the expected sprouting from nerve terminals but also induced growth from some nodes of Ranvier close to the endplate region. This finding shows that nerve degeneration is not essential for nodal sprouting and supports the hypothesis that inactive muscle liberates a motor nerve growth factor.     

Botulinum toxin treatment of children with cerebral palsy — a short review of different injection techniques

The intramuscular application of botulinum toxin type A (BoNT/A) has emerged to be an established treatment option to reduce muscular hyperactivity due to spasticity in children with cerebral palsy. Accurate injection is a prerequisite for efficient and safe treatment with BoNT/A. So far, treatment procedures have not been standardized. This paper is a short review of different injection techniques, i.e., manual needle placement as well as guidance by electromyography, electrical stimulation, and ultrasound. Advantages and disadvantages of the different injection techniques are discussed with a focus on needle positioning within the targeted muscle, injection close to the neuromuscular junction and diffusion of BoNT/A within the target muscles and through fascia. The additional information gained by each injection technique is weighed in terms of the clinical impact for children with cerebral palsy.     

Serial SFEMG studies of orbicularis oculi muscle after the first administration of botulinum toxin

Serial single fiber electromyography (SFEMG) examinations of orbicularis oculi muscle in patients with blepharospasm or hemifacial spasm treated with botulinum toxin injections were performed. The aim of the study was to evaluate the impairment of neuromuscular transmission, to follow reinnervation after botulinum toxin administration and to find out whether there was a relationship between SFEMG parameters and clinical symptoms. Examinations were performed before injection, during early and late remission of symptoms, and after recurrence of the involuntary movement. Severe impairment of neuromuscular transmission, as revealed by increased jitter and increased presence of abnormal potential pairs and pairs with blocking, was found in early remission, but fiber density remained unchanged when compared with pretreatment values. In late remission, increased fiber density was registered for the first time. The recurrence of involuntary movements was related to the further increase of fiber density and tendency to normalization of jitter parameters. The study therefore suggests that formation of new neuromuscular junctions and their functional maturation is responsible for muscle recovery after botulinum toxin administration.     

Longitudinal neurophysiological assessment of intramuscular type-A botulin toxin in healthy humans

The aim of this study is to assess the neurophysiological abnormalities of type A botulin toxin-infiltrated human muscle, and their evolution over time. Seried cMAP measurements, 3 and 20 Hz repetitive nerve stimulation, EMG, SFEMG over 3 months from toxin injection. Our findings consist in lack of decrement with 3 Hz repetitive nerve stimulation and facilitation with 20 Hz repetitive nerve stimulation; progressive increasing of jitter; early appearance of fibrillations; small and short motor unit action potential in the first 3 weeks, followed by increasing of MUAP amplitude and duration, with polyphasic morphology. Although claimed as highly specific and sensible, neuromuscular junction facilitation is an inconstant finding in human botulism. Therefore, lack of neuromuscular junction facilitation cannot exclude a diagnosis of botulism. Our findings are compatible with a process of acute denervation followed by distal reinnervation, favored by terminal nerve sprouting.     

Antagonism of the paralysis produced by botulinum toxin in the rat. The effects of tetraethylammonium, guanidine and 4-aminopyridine.

The injection of botulinum toxin type A into the hind-leg of adult rats causes complete paralysis of the leg lasting for several weeks. In the extensor digitorum longus (EDL) muscle transmitter release is reduced to a level of less than 1% of normal. Tetraethylammonium (TEA) and guanidine in concentrations of about 3 mM restore, in EDL muslces in vitro, neuromuscular transmission to about the normal level, provided that the external calcium concentration is 4 mM or higher. 4-Aminopyridine (4-AP) has similar restorative effect but is about 20-30 times more potent. Unlike TEA and guanidine, 4-AP is effective when the ambient calcium concentration is 2 mM; this drug is therefore also active in vivo. The intravenous injection of 4-AP (5 mg/kg body weight) restores neuromuscular transmission from complete paralysis by botulinum toxin to a normal level as shown by the recording of almost normal twitch and tetanic tensions in the EDL muscle. In rats paralysed by a lethal dose of botulinum toxin, the intraperitoneal administration of 4-AP restores general motor activity, the effect lasting 1-2 hours. A study of the effects of these drugs on spontaneous and evoked transmitter release suggests that all three compounds increase the level of free calcium inside the nerve terminals. In botulinum poisoning the transmitter release mechanism appears to be intact, but a reduced sensitivity to calcium has been shown (Cull-Candy et al. 1976), and this could explain why the drugs restore evoked transmitter release in botulinum poisoning.     

Scaffoldless tissue-engineered nerve conduit promotes peripheral nerve regeneration and functional recovery after tibial nerve injury in rats

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fi-broblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, re-spectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promot-ed nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.     

实验性糖尿病大鼠的单纤维肌电图研究

目的:分析链脲佐菌素(STZ)诱导的糖尿病大鼠坐骨神经的神经传导速度和腓肠肌单纤维肌电图的特点,评价两种检查方法对糖尿病多发性神经病早期诊断的价值.方法:健康雄性SD大鼠经腹腔注射STZ60mg穔g-1诱导成1型糖尿病大鼠模型(糖尿病组),在注射STZ后4、6、8、10和12周进行坐骨神经神经传导检查和腓肠肌单纤维肌电...     

Terminal sprouting is not responsible for enhanced transmitter release at disused neuromuscular junctions of the rat

Chronic block of nerve-muscle activity is known to induce sprouting of motor nerve terminals and to enhance transmitter release at the neuromuscular junction. Increased transmitter release has been assumed to be a physiological correlate of disuse-induced sprouting of nerve terminals. We examined this assumption in the rat extensor digitorum longus muscle following chronic conduction block of the sciatic nerve with TTX. The minimal period of nerve block required for the expression of terminal sprouting was 3 d, whereas transmitter release, measured by the quantal analysis of end-plate potentials, was already enhanced within 24 hr of nerve block. Following 6 d of nerve block, sprouting was observed in about 35% of the motor nerve terminals examined. Under this condition, the total length of individual terminals was significantly greater in the terminals with sprouts than those without sprouts. However, enhancement of transmitter release occurred uniformly at these junctions regardless of the presence or absence of terminal sprouts. Also, transmitter release enhanced by nerve block for 2 d remained elevated for at least 4 d even after resumption of nerve activity without the formation of terminal sprouts. It is concluded that terminal sprouting and increased transmitter release induced in disused neuromuscular junctions are not causally related and that the signals for inducing these 2 events are at least quantitatively different.     

Importance of pathway formation for nodal sprout production in partly denervated muscles

Experiments were carried out to investigate possible factors controlling nodal sprout growth in partly denervated mouse gluteal muscles. Pretreatment of the muscle with botulinum toxin for up to 20 days, which produces denervation-like changes and elicits terminal⁵ and nodal¹⁰ sprouting, increased reinnervation by terminal sprouting after partial denervation but did not alter the rate of reinnervation by nodal sprouts. This implies that nodal sprout growth is not limited by the development of an adequate growth stimulus from denervated muscle. A distro-proximal gradient of degeneration was observed in denervated intramuscular nerves in the electron microscope, suggesting that nodal sprout growth may be modulated by the availability of endoneurial pathways sufficiently degenerated to permit reinnervation by nodal sprouts, although the initial outgrowths from nodes of Ranvier may appear in response to a growth stimulus from regenerated.