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Biomedical subjects

L Greensmith

Publications and source records attributed to L Greensmith.

35 records · Page 2Linked to original sources
PubMed1997

A simple method for local delivery of various substances to the rat neuromuscular system.

We report here a simple method for the local delivery of various substances to the neuromuscular system in developing and adult rats. This method permits continuous treatment of tissues with a compound over a period of days. Alternative drug delivery systems are unsuitable in neonates. Osmotic pumps are too large and repetitive injections damage the tissues in neonatal rats. Our delivery system provides an adaptable means by which we can directly apply substances in various concentrations in implants of differing sizes. Substances are incorporated into flexible, non-toxic silicone rubber. Strips are cut from the rubber for implantation alongside the muscle or nerve in the anaesthetised animal. The size of the strip is tailored to the length of the muscle or nerve requiring the treatment. Release of the substance from the implant occurs over a period of days and if a longer period of treatment is required, the initial strip can be replaced with a second and even a third implant. We have tested the effects of the substances applied in this manner both physiologically, by examination of muscle function, and morphologically, by muscle histology and retrograde labelling of motoneurones. We have successfully used this method for the application of various groups of substances, including neurotoxins, channel blockers (K+, Ca2+ and Cl-), calcium-chelating agents, protease inhibitors and ionic salts.

Animals↗
PubMed1996

Transient disruption of nerve-muscle interaction shortly after birth permanently alters the development of the rat soleus muscle.

Transient paralysis of the rat soleus muscle shortly after birth leads to a permanent loss of motoneurones as revealed by retrograde labelling. Here we show that this loss of motoneurones is reflected in a reduction in the number of motor units. Soleus muscles in normal adult rats were found to have 27 (+/-0.6 S.E.M., n = 9) motor units. However, in muscles which had been treated with alpha-bungarotoxin (BTX) at birth and 3 days of age, causing paralysis lasting for 6-8 days, only 15(+/-0.6 S.E.M., n = 5) motor units remain. The effects of paralysis on the ability of the adult soleus muscle to develop force was also tested. Following treatment with a single BTX implant at birth, causing paralysis for 2-3 days, soleus muscles develop less tension (73.7% +/- 4.5 S.E.M., n = 8) and weigh less (88.2% +/- 3.8 S.E.M., n = 13) than their unoperated controls. This loss of muscle force is caused by a loss of muscle fibres, which in muscles that had been paralysed at birth was 81.4% (+/-4.1 S.E.M., n = 5) of control. Prolonging the duration of paralysis led to a greater reduction in force production, weight and the number of muscle fibres. Those muscles which had been paralysed at birth also took longer to relax during single twitch contractions. In addition, whereas normal soleus muscles contain around 20% of muscle fibres that do not react with antibodies to slow myosin HC, in soleus muscles paralysed at birth, 100% of the fibres reacted with this antibody. This study shows that disruption of neuromuscular interaction for a brief period after birth leads to a loss of motoneurones and a permanent impairment of muscle function.

Animals↗
PubMed1996

Induction of transmitter release at the neuromuscular junction prevents motoneuron death after axotomy in neonatal rats.

Motoneurons to rat hindleg muscles die after neonatal nerve injury. Here we show that increasing transmitter release of motor nerve terminals by treatment with 4-aminopyridine, prior to nerve injury at three days, reduces the extent of motoneuron death. Retrograde labelling of soleus motoneurons was carried out in 10-week-old animals that had their sciatic nerve crushed on one side when they were three days old. Only 20% (+/- 4.2 S.E.M.) of the motoneurons survived the nerve injury. A group of animals similarly injured at three days had their calf muscles treated with 4-aminopyridine at birth, prior to nerve injury. In these animals a significantly higher percentage (51 +/- 6.6% S.E.M.) of soleus motoneurons survived. In order to assess the proportion of surviving alpha-motoneurons only, the number of motor units in both the soleus and extensor digitorum longus muscles was established by following the stepwise increments of twitch tension in response to increasing intensity of stimulation of the respective motor nerve. After nerve injury at three days only 18% (+/- 4.1% S.E.M.) of motor units to soleus and 28.5% (+/- 4.9% S.E.M.) to extensor digitorum longus survived and were able to reinnervate their respective muscles. If the nerve injury was preceded by local application of 4-aminopyridine, then the number of motor units present in the reinnervated muscles was significantly greater, so that in soleus 52.7% (+/- 5.4% S.E.M.) and in extensor digitorum longus 52.1% (+/- 2.4% S.E.M.) of motor units were present. This increase of motoneuron survival was reflected in a smaller weight loss and in a better restoration of force production by the pretreated as compared to untreated muscles on reinnervation after nerve injury. It is suggested that enhancing transmitter release from nerve endings in neonatal animals induces the motoneuron to become more resistant to nerve injury.

4-Aminopyridine↗
PubMed1996

Motoneurone survival: a functional approach.

The role of the target in the survival of developing motoneurones is discussed. The contribution of neurotrophic factors is re-evaluated in view of the following: (1) motoneurone numbers are affected only slightly in transgenic mice following deletions of genes coding for neurotrophins or their receptors; and (2) continued treatment with neurotrophins fails to achieve long-term motoneurone survival. Evidence that motoneurone survival depends on the induction of transmitter release initiated by contact between nerve and muscle is presented. The release of transmitter and the ensuing retraction of many axon branches transforms the motoneurone from a growing cell into a transmitting cell. It is suggested that only when motoneurones have undergone this transition can they survive within the mature CNS.

Animals↗
PubMed1996

Stabilizing neuromuscular contacts increases motoneuron survival after neonatal nerve injury in rats.

Following sciatic nerve crush at birth the rat soleus muscle is rendered permanently weak. This reduction in muscle force is caused by the loss of a proportion of its motoneurons. Furthermore, motoneurons that survive and reach the muscle fail to reoccupy a sufficient number of denervated muscle fibres to compensate for the loss of neurons. Both the loss of motoneurons and poor reinnervation may be due to the inability of the regenerating axons to establish and maintain neuromuscular contacts. Application of leupeptin, an inhibitor of a calcium-activated neutral protease and some serine proteases, is known to help in the maintenance of neuromuscular contacts during development and axonal sprouting. Here we examined whether protecting new neuromuscular contacts formed between regenerating axons and denervated muscle fibres after nerve injury, would influence the survival of motoneurons and improve muscle recovery. This study shows that in muscles treated with leupeptin the reduction in weight and force output after nerve crush at birth was significantly less than in those that were untreated. Moreover, the number of motor units in the leupeptin-treated muscles was significantly higher than in untreated muscles. Thus, treating regenerating nerve terminals with leupeptin during early stages of reinnervation rescues motoneurons and improves muscle recovery.

Animals↗
PubMed1995

Effect of transient neonatal muscle paralysis on the growth of soleus motoneurones in the rat.

The postnatal growth of soleus motoneurones was studied during normal development and following transient paralysis of the soleus muscle in neonatal rats. Paralysis was achieved by implanting a silicon strip containing alpha-bungarotoxin alongside the soleus muscle in rat pups within 3-6 h of birth. The soleus muscle was completely paralysed for at least 24 h, and by 9 days neuromuscular transmission was fully restored. The soma size of normal and target-deprived soleus motoneurones was compared at intervals during the first 3 postnatal weeks and in adults, using the retrograde horseradish peroxidase technique. There was a four-fold increase in the soma area of normal motoneurons during the first 3 postnatal weeks, with the greater part of the increase occurring between 7 and 14 days. At 3 days, the distribution of soma areas was unimodal and became bimodal by 21 days. Paralysis during the first postnatal week did not significantly affect the developmental changes in motoneurone soma area or their distribution up to 3 weeks of age. Thus, motoneurones deprived of functional neuromuscular contact appear to grow normally during the early postnatal period, although previous results show that at later stages (2-3 months of age), many of these motoneurones die and the remaining cells are smaller than normal.

Age Factors↗
PubMed1995

Transient muscle paralysis in neonatal rats renders motoneurons susceptible to N-methyl-D-aspartate-induced neurotoxicity.

Paralysis of the soleus muscle in newborn rats causes a large proportion of motoneurons to die by 10 weeks of age. However, all of these neurons are still present at three to four weeks of age. We have previously shown that although nerve injury at five days does not result in any motoneuron death, it does render these neurons susceptible to the toxic effects of the glutamate agonist N-methyl-D-aspartate. Using retrograde labelling of soleus motoneurons, in this study we show that an increased susceptibility to glutamate also plays a role in the eventual death of those motoneurons which survive for three weeks after interruption of neuromuscular transmission at birth but die by 10 weeks. Treatment with dizocilpine maleate an antagonist of the N-methyl-D-aspartate receptor increased the survival of motoneurons to alpha-bungarotoxin-treated soleus muscles. By 10 weeks of age the size of motoneurons to alpha-bungarotoxin-treated soleus muscles is smaller than that of controls, but after treatment with dizocilpine maleate the sizes of motoneurons to control and treated muscles are similar. Moreover, only 55 +/- 2.7% of motoneurons to the soleus muscle paralysed at birth with alpha-bungarotoxin survive for three weeks after a single injection of N-methyl-D-aspartate at 12 days of age. This motoneuron death is due to the application of N-methyl-D-aspartate since treatment with alpha-bungarotoxin alone causes no loss of neurons at this age.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Magnesium ions reduce motoneuron death following nerve injury or exposure to N-methyl-D-aspartate in the developing rat.

Developing motoneurons can be induced to die by target deprivation and there is evidence that this cell death involves the excitotoxic effects of N-methyl-D-aspartate. Treatment with dizocilpine maleate, an antagonist of this receptor, has been shown to rescue a proportion of those motoneurons destined to die following nerve injury at birth. However, this is a relatively toxic compound. In this study we examined whether systemic treatment with magnesium sulphate, a non-competitive antagonist of the N-methyl-D-aspartate receptor which is better tolerated than dizocilpine maleate, could prevent motoneuron death. Motoneurons were induced to die either by sciatic nerve injury at birth or by nerve injury at five days followed by exposure to N-methyl-D-aspartate. The number of surviving motoneurons reinnervating the tibialis anterior and extensor digitorum longus muscles were counted using retrograde labelling. Following nerve injury at birth and treatment with magnesium sulphate, there was a small increase in the survival of injured motoneurons, although this improvement was not significant. Nerve injury at five days does not result in motoneuron death, but when followed by treatment with N-methyl-D-aspartate, only 42 +/- 2.9% of motoneurons to these flexor muscles survived. Treatment with magnesium sulphate prior to injection of N-methyl-D-aspartate significantly increased motoneuron survival, so that 67 +/- 5.8% of motoneurons survived. Thus, systemic treatment with magnesium can prevent the death of motoneurons rendered susceptible to the excitotoxic effects of N-methyl-D-aspartate by nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Blocking of NMDA receptors during a critical stage of development reduces the effects of nerve injury at birth on muscles and motoneurones.

Blocking of NMDA receptors during a critical stage of development reduces the effects of nerve injury at birth on muscles and motoneurones. Injury to the sciatic nerve at birth causes many motoneurones to soleus and extensor digitorum longus (EDL) muscles of rats to die. This is reflected in a reduction of motor units in these muscles. In the soleus only 4 (12.3%) motor units remain while 10 (24.3%) remain in the EDL, showing that soleus alpha motoneurones are more sensitive to nerve injury at birth. Treatment with MK-801, an NMDA receptor blocker, rescues a proportion of motor units in both muscles, so that in the soleus 11 (36%) and in the EDL 17 (42%) of motor units survive. This loss of motor units results in muscle weakness and a reduction in force of both muscles. Treatment with MK-801 reduces the effect of nerve injury, so that muscles of treated animals are stronger and weigh more. Cross-sectional area and muscle fibre number in EDL muscles were assessed and found to be dramatically reduced after nerve injury at birth, so that the area was 20% of control, with only 13% of fibres remaining. Moreover the majority of the remaining EDL muscle fibres which are normally fast are converted into slow type I fibres, with 68% of fibres expressing slow myosin compared with 3% in control EDL muscles. In animals treated with MK-801 only 47% of muscle fibres are lost after nerve injury at birth, hence the area of the muscle is greater (51% of control). The change of muscle phenotype induced by nerve injury is prevented and the muscle fibre composition resembles that of normal EDL muscles in that 4% of muscle fibres express slow myosin compared with 3.5% in control EDL muscles. Thus, blocking NMDA receptors with MK-801 shortly after nerve injury at birth reduces the loss of motor units and this is directly reflected in an improved performance of the affected muscles.

Animals↗
PubMed1995

Possible strategies for treatment of SMA patients: a neurobiologist's view.

This paper discusses possible strategies that might prevent or alleviate muscle weakness of SMA patients and hence improve their condition. The strategies discussed are as follows. (1) Prevention of motoneurone death. To achieve this two main approaches have been applied. Firstly, trophic factors have been used to prevent motoneurone death after nerve injury and clinically in diseases such as motoneurone disease. The results of these attempts will be described. Secondly, the possibility that injured motoneurones die as a result of the excitotoxic effects of the excitatory transmitter glutamate will be explored. Evidence will be presented which indicates that blocking glutamate receptors can rescue injured motoneurones from death. (2) Replacement of lost motoneurones by embryonic grafts. Motoneurones from grafts of embryonic spinal cord have been shown to survive in the adult spinal cord and are able to reinnervate skeletal muscles. The potential and practical problems of this approach will be discussed. (3) Expansion or motor unit territory of surviving motoneurones. Such an expansion of the territory occupied by individual motor units can be achieved by encouraging sprouting and ensuring that the newly formed connections between the motoneurone and muscle fibres are maintained, so that individual motor units are capable of developing more force. Strategies to achieve such an expansion of motor unit territory will be described. Finally, combinations of some of these approaches are considered.

Humans↗
PubMed1994

Blockade of N-methyl-D-aspartate receptors by MK-801 (dizocilpine maleate) rescues motoneurones in developing rats.

In rats following nerve injury at birth a large proportion of motoneurones to the soleus muscle dies. Blocking of N-methyl-D-aspartate (NMDA) receptors by MK-801 (dizocilpine maleate) for 12 days after nerve injury at birth leads to rescue of a proportion of motoneurones destined to die. Retrograde labelling of soleus motoneurones shows that 6-8 weeks after crushing the sciatic nerve in one hindlimb, only 10.9 +/- 2.3% of the motoneurones have survived. In animals treated with an NMDA receptor blocker MK-801 (2 mg/kg i.p., from birth to 12 days old) 50.6 +/- 3.8% of soleus motoneurones survived. This neuroprotective effect of MK-801 was dose dependant, since after treatment with lower doses (0.5 mg/kg; 1 mg/kg) fewer motoneurones survived (13.7% and 34.5%, respectively). To assess the effect of treatment with MK-801 on survival of alpha-motoneurones only, the number of soleus motor units was established physiologically. After nerve injury alone only 4.2 +/- 1.2 of the 29-30 soleus motor units were present, while in animals treated with MK-801 (2 mg/kg) 14 +/- 1.5 motor units were identified. The neuroprotective effect of MK-801 was not confined to soleus motoneurones but was also apparent on motoneurones to the extensor digitorum longus (EDL). In untreated EDL muscles of the 40 motor units only 5.5 +/- 1.7 motor units survived neonatal nerve injury and this number increased to 18 +/- 2.6 after treatment with MK-801. The neuroprotective effect of MK-801 was apparent regardless of whether the nerve lesion was carried out close to or far from the soleus muscle.

Animals↗
PubMed1994

Nerve injury increases the susceptibility of motoneurons to N-methyl-D-aspartate-induced neurotoxicity in the developing rat.

If the sciatic nerve is crushed in neonatal rats, a large proportion of motoneurons die, but the same injury inflicted at five days of age results in little, if any, motoneuron death. However, these motoneurons are rendered susceptible to the excitotoxic effects of the glutamate agonist, N-methyl-D-aspartate. Retrograde labelling of soleus motoneurons after nerve crush at five days of age, followed by treatment with N-methyl-D-aspartate seven days later, shows that only 36 +/- 7.5% of motoneurons have survived. If the motoneurons are allowed to reinnervate their target, and N-methyl-D-aspartate is applied three weeks after the nerve injury, no motoneuron death is observed. Furthermore, adult motoneurons remain resistant to the toxic effects of N-methyl-D-aspartate, even after nerve injury. These results indicate that glutamate, the main excitatory neurotransmitter in the developing spinal cord, may be involved in the motoneuron death that occurs following nerve injury during early postnatal development.

Animals↗
PubMed1993

Motoneurons destined to die are rescued by blocking N-methyl-D-aspartate receptors by MK-801.

Motoneurons to soleus muscle die if their axons are injured at birth. We tested the possibility that their death may be caused by toxic effects of excitatory transmitters such as glutamate. Animals that had their sciatic nerves crushed at birth were treated either with a blocker of the N-methyl-D-aspartate receptor, dizocilpine maleate (MK-801), or saline. The number of surviving soleus motoneurons and motor units was assessed 60-90 days later. Treatment of rats with MK-801 rescued a large proportion of injured motoneurons destined to die. Moreover, the weight loss of soleus muscles seen after nerve injury at birth was reduced in animals treated with MK-801. These results suggest that motoneurons axotomized at birth are unable to withstand the excitotoxic effects of glutamate and die. Blocking glutamate receptors in conditions where motoneuron loss occurs could be an effective way of rescuing them.

Animals↗
PubMed1993

Possible consequences of disruption of neuromuscular contacts in early development for motoneurone survival.

Motoneurones are known to die (1) during embryonic development (naturally occurring cell death), (2) early in postnatal development after axonal injury, and (3) as a consequence of disease such as SMA. Interactions with the target emerges as an important factor for survival of developing motoneurones. The evidence for the target dependence od of developing motoneurones will be presented and the mechanisms by which the muscle may regulate motoneurone survival discussed. Results that argue for the following proposal will be given: with maturation of the CNS motor activity in all mammals increases as do the functional demands on the motoneurones. The target muscle's role is to induce changes in the motoneurone to make it competent to respond to increased amounts of glutamate from excitatory inputs and thus allow it to carry out the tasks associated with its increased activity. A failure of the muscle to induce these changes in the motoneurone's phenotype in time may lead to motoneurone death. In addition new approaches that could (1) improve motoneurone survival, and (2) use embryonic grafts to replace the lost cells will be discussed.

Animals↗
PubMed1992

Alterations of nerve-muscle interaction during postnatal development influence motoneurone survival in rats.

The effect of temporary paralysis of rat soleus muscles shortly after birth on motoneurone survival was studied using retrograde labelling with HRP. Following a single application of alpha-bungarotoxin (alpha-BTX) at birth the muscles were paralysed for 24-48 h. The number of HRP-labelled motoneurones in the treated ventral horn of the spinal cord in 10-week-old rats decreased to 63.7% (+/- 2.7 S.E.M.) of the control ventral horn. This motoneurone loss occurs relatively late after alpha-BTX application, for in animals examined at 3-4 weeks of age, the number of labelled motoneurones in the treated ventral horn was not reduced. When paralysis of the soleus muscles was extended to 6-8 days by application of an additional alpha-BTX implant, then by 10 weeks of age only 34.7% (+/- 1.5 S.E.M.) of soleus motoneurones were present. Thus, prolonging the duration of paralysis reduced the number of surviving motoneurones. Furthermore, the mean area of motoneurones that survived alpha-BTX treatment was smaller than that of controls. There was also a shift in the size distribution of the motoneurones in that there was a relative increase in the proportion of small motoneurones. This finding is similar to observations on motoneurone sizes after neonatal nerve injury. Thus, interruption of neuromuscular interaction during early postnatal development causes many motoneurones to die, and in addition alters the size distribution of the remaining cells.

Animals↗
PubMed1991

Neuromuscular contacts in the developing rat soleus depend on muscle activity.

Neuromuscular transmission of the rat soleus muscle was interrupted by blocking the response of the postsynaptic membrane with alpha-bungarotoxin (alpha-BTX) at two different stages of postnatal development, i.e., at birth and at 10 days. The effect of this treatment on the maintenance of synaptic contacts was studied using histological and electrophysiological criteria. Following treatment at birth fewer muscle fibres were polyneuronally innervated 5-7 days later. After this initial loss of synaptic contacts, the subsequent rate of synapse elimination was slower than in control animals, so that even at 3 weeks muscles treated with alpha-BTX at birth had higher levels of polyneuronal innervation than their unoperated controls. Thus, interference with the response of the postsynaptic membrane at birth has prolonged effects on synaptic development. In muscles treated with alpha-BTX at 10 days the elimination of polyneuronal innervation was arrested and more neuromuscular contacts preserved.

Animals↗